CN100537764C

CN100537764C - Metabolic protein for weed-control agent, its gene and application thereof

Info

Publication number: CN100537764C
Application number: CNB028256425A
Authority: CN
Inventors: 中岛宽树; 椋本藤夫; 高石昌直
Original assignee: Sumitomo Chemical Co Ltd
Current assignee: Sumitomo Chemical Co Ltd
Priority date: 2001-10-19
Filing date: 2002-10-17
Publication date: 2009-09-09
Anticipated expiration: 2022-10-17
Also published as: CN1894408A

Abstract

For example the invention provides, coding is selected from the DNA of the metabolic protein for weed-control agent of following protein group.This DNA can for example be used to produce herbicide resistant plants.＜protein group〉a kind of protein, it is included in SEQ ID NO:1,2,3,108,159,160,136,137,138,215,216,217,218,219,220, the aminoacid sequence of expression in 221,222,223 or 224, a kind of protein, it has in the presence of the electron transport system that comprises electron donor formula (II) compound is converted into the ability of formula (III) compound, and comprises and in SEQ ID NO:1,2,3,108,159,136,137,138,215,216,217,218,219,220,221, in 222,223 or 224 the aminoacid sequence of any one expression have at least 80% sequence identity aminoacid sequence or with in SEQ ID NO:160,215, the aminoacid sequence of any one expression has the aminoacid sequence of at least 90% sequence identity in 216,218,222 or 224.

Description

Metabolic protein for weed-control agent, its gene and application thereof

Technical field

The present invention relates to have the protein (metabolic protein for weed-control agent) of metabolism herbicidal compound ability, its gene and application thereof.

Background technology

Weedicide is the diluent utilization with requirement when using.There is excessive remaining situation.Also exist the weedicide of wherein using to use the situation that moments later remains in soil or the plant residue at it.At first, suppose the security of these weedicides on inspection, these a spot of surplus solutions or resistates are done the little influence of deposits yields to environment or cultivation after this.Yet,, so for example can carry out the processing of above-mentioned surplus solution of passivation or resistates as required if exist the herbicidal compound that will comprise to be converted into a kind of method of compound of low weeding activity.

In addition, in the situation of using weedicide, exist the weeds that are difficult to cultivated plant and allied species to distinguish optionally only to control the situation of weeds.Thereby, need exploitation to give the novel method of target plant herbicide resistance.

Summary of the invention

In this case, inventor's further investigation, the result has been found that it causes of the present invention finishing by the herbicidal compound of proporphyrinogen oxidase (hereinafter, being sometimes referred to as " PPO ") inhibition type being converted into the compound that hangs down weeding activity with the particular proteins reaction.

That is, the invention provides:

1. coding herbicide metabolism proteic DNA, wherein said protein is selected from:

(A1) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:1;

(A2) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:2;

(A3) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:3;

(A4) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:108;

(A5) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor,

And comprise and at SEQ ID NO:1, SEQ ID NO:2, the aminoacid sequence of any one expression has the aminoacid sequence of at least 80% sequence identity among SEQ ID NO:3 or the SEQ ID NO:108;

(A6) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise nucleotide sequence coded aminoacid sequence, described nucleotide sequence be coded in SEQ ID NO:1, SEQ ID NO:2, the nucleotide sequence of the aminoacid sequence of any one expression has at least 80% sequence identity among SEQ ID NO:3 or the SEQ ID NO:108;

(A11) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:159;

(A12) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:160;

(A13) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:136;

(A14) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:137;

(A15) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:138;

(A16) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:215;

(A17) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:216;

(A18) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:217;

(A19) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:218;

(A20) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:219;

(A21) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:220;

(A22) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:221;

(A23) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:222;

(A24) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:223;

(A25) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:224;

(A26) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise and at SEQ ID NO:159, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:220, among SEQ ID NO:221 or the SEQ ID NO:223 aminoacid sequence of any one expression have at least 80% sequence identity aminoacid sequence or with at SEQ ID NO:160, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:218, the aminoacid sequence that the aminoacid sequence of any one expression has at least 90% sequence identity among SEQ ID NO:222 or the SEQ ID NO:224.

(A27) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise nucleotide sequence coded aminoacid sequence, described nucleotide sequence be coded in SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQID NO:220, SEQ ID NO:221, SEQ ID NO:222, the nucleotide sequence of the aminoacid sequence of any one expression has at least 90% sequence identity among SEQ ID NO:223 or the SEQ IDNO:224; With

(A28) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise by the coded aminoacid sequence of DNA that can pass through polymerase chain reaction (PCR) amplification, primer that is included in the nucleotide sequence of any one expression among the SEQ ID NO:124 to 128 and the primer that is included in the nucleotide sequence of representing among the SEQ ID NO:129 are used in described polymerase chain reaction, and template is abortion within the first month of pregnancy look streptomycete (Streptomyces phaeochromogenes), brick-red streptomycete (Streptomycestestaceus), do not produce look streptomycete (Streptomyces achromogenes), ash brown streptomycete (Streptomyces griseofuscus), hot lightskyblue streptomycete (Streptomycesthermocoerulescens), black walnut streptomycete (Streptomyces nogalater), Tianjin island chain mould (Streptomyces tsusimaensis), pelletizing produces look streptomycete (Streptomycesglomerochromogenes), olive produces look streptomycete (Streptomyces olivochromogenes), decorative chains mould (Streptomyces ornatus), streptomyces griseus (Streptomyces griseus), wool streptomycete (Streptomyces lanatus), three damp streptomycetes (Streptomyces misawanensis), pale asphyxia streptomycete (Streptomyces pallidus), the rose streptomycete (Streptomycesroseorubens) that reddens, ground, Shandong streptomycete (Streptomyces rutgersensis), the chromosomal DNA of Regensburg streptomycete (Streptomyces steffisburgensis) or Ta Shi saccharopolyspora strain (Saccharopolyspora taberi);

2. the DNA that comprises nucleotide sequence, described nucleotide sequence is selected from:

(a1) nucleotide sequence of in SEQ ID NO:6, representing;

(a2) nucleotide sequence of in SEQ ID NO:7, representing;

(a3) nucleotide sequence of in SEQ ID NO:8, representing;

(a4) nucleotide sequence of in SEQ ID NO:109, representing;

(a5) nucleotide sequence of in SEQ ID NO:139, representing;

(a6) nucleotide sequence of in SEQ ID NO:140, representing;

(a7) nucleotide sequence of in SEQ ID NO:141, representing;

(a8) nucleotide sequence of in SEQ ID NO:142, representing;

(a9) nucleotide sequence of in SEQ ID NO:143, representing;

(a10) nucleotide sequence of in SEQ ID NO:225, representing;

(a11) nucleotide sequence of in SEQ ID NO:226, representing;

(a12) nucleotide sequence of in SEQ ID NO:227, representing;

(a13) nucleotide sequence of in SEQ ID NO:228, representing;

(a14) nucleotide sequence of in SEQ ID NO:229, representing;

(a15) nucleotide sequence of in SEQ ID NO:230, representing;

(a16) nucleotide sequence of in SEQ ID NO:231, representing;

(a17) nucleotide sequence of in SEQ ID NO:232, representing;

(a18) nucleotide sequence of in SEQ ID NO:233, representing;

(a19) nucleotide sequence of in SEQ ID NO:234, representing;

(a20) a kind of nucleotide sequence, the aminoacid sequence of its coded protein, described protein has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, described nucleotide sequence with at SEQ ID NO:6, SEQ ID NO:7, the nucleotide sequence of any one expression has at least 80% sequence identity among SEQ IDNO:8 or the SEQ ID NO:109; With

(a21) a kind of nucleotide sequence, the aminoacid sequence of its coded protein, described protein has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, described nucleotide sequence with at SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:225, SEQID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ IDNO:230, SEQ ID NO:231, SEQ ID NO:232, the nucleotide sequence of any one expression has at least 90% sequence identity among SEQ ID NO:233 or the SEQ IDNO:234;

3. according to above-mentioned 1 DNA, it comprises the nucleotide sequence of the aminoacid sequence of code for said proteins, and wherein to use be that codon uses in ± 4% the scope and the GC content of described nucleotide sequence is at least 40% and at the most 60% in from the gene of the host cell species of introducing DNA to the codon in described nucleotide sequence;

4. be included in the DNA of the nucleotide sequence of representing among the SEQ ID NO:214;

5. be included in the DNA of the nucleotide sequence of representing among the SEQ ID NO:368;

6. be included in the DNA of the nucleotide sequence of representing among the SEQ ID NO:393;

7. DNA, the DNA that wherein has a nucleotide sequence of coding born of the same parents inner cell organ encoding transport signals sequence is connected the upstream according to DNA in above-mentioned 1 the frame;

8. DNA, wherein according to above-mentioned 1 DNA and the promotor that in host cell, works operationally (operably) be connected;

9. comprise carrier according to above-mentioned 1 DNA;

10. method that produces carrier, it comprises will insert the step of the carrier that can duplicate according to above-mentioned 1 DNA in the host;

11. a transformant wherein will be introduced host cell according to above-mentioned 1 DNA;

12. the transformant according to above-mentioned 11, wherein said host cell are microorganism cells or vegetable cell;

13. a method that produces transformant, it comprises and will introduce the step of host cell according to above-mentioned 1 DNA;

14. a generation has the method for protein that formula (II) compound is converted into the ability of formula (III) compound, described method comprises cultivation according to above-mentioned 11 transformant with reclaim the described proteinic step that produces;

15. the DNA according to above-mentioned 1 has application in the protein of the ability that formula (II) compound is converted into formula (III) compound in production;

16. a method that gives the plant herbicide resistance, described method comprise according to above-mentioned 1 DNA introduced plant cell and the step expressed therein;

17. polynucleotide, its have according to the partial nucleotide sequence of above-mentioned 1 DNA or with described partial nucleotide sequence complementary nucleotide sequence;

18. one kind is detected coding and has the method for protein DNA that formula (II) compound is converted into the ability of formula (III) compound, described method comprise detection in hybridization with the step of the DNA of probe hybridization, its use according to above-mentioned 1 DNA or according to above-mentioned 17 polynucleotide as probe;

19. one kind is detected coding and has the method for protein DNA that formula (II) compound is converted into the ability of formula (III) compound, described method comprises detection in the step with the DNA that increases in according to above-mentioned 17 the polymerase chain reaction of polynucleotide as primer;

20. the method according to above-mentioned 19, the wherein at least a polynucleotide that are selected from the group of the polynucleotide composition that is included in the nucleotide sequence of any one expression among the SEQ IDNO:124 to 128 and are included in the nucleotide sequence that shows among the SEQ ID NO:129 in the primer;

21. one kind obtains coding and has the method for protein DNA that formula (II) compound is converted into the ability of formula (III) compound, described method comprises the step that reclaims by the DNA that detects according to above-mentioned 18 or 19 method;

22, a kind of screening contains coding and has the method for cell of protein DNA that formula (II) compound is converted into the ability of formula (III) compound, and described method comprises by detect the step of described DNA from the test cell according to above-mentioned 18 or 19 method;

23. a herbicide metabolism albumen, it is selected from:

(A5) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise and at SEQ ID NO:1, SEQ ID NO:2, the aminoacid sequence that the aminoacid sequence of any one expression has at least 80% sequence identity among SEQ ID NO:3 or the SEQ ID NO:108;

(A6) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise by nucleotide sequence coded aminoacid sequence, described nucleotide sequence be coded in SEQ ID NO:1, SEQ IDNO:2, the nucleotide sequence of the aminoacid sequence of any one expression has at least 80% sequence identity among SEQ ID NO:3 or the SEQ ID NO:108;

(A26) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise and at SEQ IDNO:159, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ IDNO:217, SEQ ID NO:219, SEQ ID NO:220, among SEQ ID NO:221 or the SEQ IDNO:223 aminoacid sequence of any one expression have at least 80% sequence identity aminoacid sequence or with at SEQ ID NO:160, SEQ ID NO:215, SEQ ID NO:216, SEQ IDNO:218, the aminoacid sequence that the aminoacid sequence of any one expression has at least 90% sequence identity among SEQ ID NO:222 or the SEQ ID NO:224;

(A27) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise by nucleotide sequence coded aminoacid sequence, described nucleotide sequence be coded in SEQ ID NO:159, SEQ IDNO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ IDNO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ IDNO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, the nucleotide sequence of the aminoacid sequence of any one expression has at least 90% sequence identity among SEQ IDNO:223 or the SEQ ID NO:224; With

(A28) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise by the coded aminoacid sequence of DNA that can pass through polymerase chain reaction (PCR) amplification, primer that is included in the nucleotide sequence of any one expression among the SEQ ID NO:124 to 128 and the primer that is included in the nucleotide sequence of representing among the SEQ ID NO:129 are used in described polymerase chain reaction, and template is an abortion within the first month of pregnancy look streptomycete, brick-red streptomycete, do not produce the look streptomycete, the brown streptomycete of ash, hot lightskyblue streptomycete, black walnut streptomycete, Tianjin island chain mould, pelletizing produces the look streptomycete, olive produces look streptomycete, decorative chains mould, streptomyces griseus, the wool streptomycete, three damp streptomycetes, pale asphyxia streptomycete, the rose streptomycete that reddens, ground, Shandong streptomycete, the chromosomal DNA of Regensburg streptomycete or Ta Shi saccharopolyspora strain;

24. the proteic antibody of identification herbicide metabolism, described herbicide metabolism albumen is selected from the group of being made up of the following formula material:

(A5) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise and at SEQ ID NO:1, SEO ID NO:2, the aminoacid sequence that the aminoacid sequence of any one expression has at least 80% sequence identity among SEQ ID NO:3 or the SEQ ID NO:108;

25. one kind is detected the proteic method of herbicide metabolism, described method comprises:

(1) with sample and the step that contact of the described proteinic antibody of identification with

(2) detect the step of complex body that produced by described contact, described protein and described antibody, wherein said protein is selected from the group of being made up of the following formula material:

26. analyze or detection kit for one kind, it comprises the antibody according to above-mentioned 24;

27. the DNA of the ferredoxin of encoding, described ferredoxin is selected from the group of being made up of the following formula material:

(B1) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:12;

(B2) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:13;

(B3) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:14;

(B4) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:111;

(B5) comprise with at SEQ ID NO:12, SEQ ID NO:13, the aminoacid sequence of any one expression has the ferredoxin of the aminoacid sequence of at least 80% sequence identity among SEQ ID NO:14 or the SEQID NO:111;

(B6) comprise ferredoxin by nucleotide sequence coded aminoacid sequence, wherein said nucleotide sequence be coded in SEQ ID NO:12, SEQ ID NO:13, the nucleotide sequence of the aminoacid sequence of any one expression has at least 90% sequence identity among SEQ ID NO:14 or the SEQID NO:111;

(B7) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:149;

(B8) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:150;

(B9) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:151;

(B10) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:152;

(B11) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:153;

(B12) a kind of ferredoxin, its comprise with at SEQ ID NO:149, SEQ IDNO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:245, SEQ IDNO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, among SEQ IDNO:251 or the SEQ ID NO:253 aminoacid sequence of any one expression have at least 80% sequence identity aminoacid sequence or with at SEQ ID NO:150, the aminoacid sequence that the aminoacid sequence of any one expression has at least 90% sequence identity among SEQ ID NO:252 or the SEQ IDNO:254;

(B13) a kind of ferredoxin, it comprises by nucleotide sequence coded aminoacid sequence, described nucleotide sequence be coded in SEQ ID NO:149, SEQ ID NO:150, SEQ IDNO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:245, SEQ IDNO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ IDNO:251, SEQ ID NO:252, the nucleotide sequence of the aminoacid sequence of any one expression has at least 90% sequence identity among SEQ ID NO:253 or the SEQ ID NO:254;

(B14) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:245;

(B15) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:247;

(B16) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:248;

(B17) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:249;

(B18) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:250;

(B19) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:251;

(B20) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:252;

(B21) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:253; With

(B22) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:254;

28. comprise the DNA of nucleotide sequence, described nucleotide sequence is selected from the group of being made up of following substances:

(b1) nucleotide sequence of in SEQ ID NO:15, representing;

(b2) nucleotide sequence of in SEQ ID NO:16, representing;

(b3) nucleotide sequence of in SEQ ID NO:17, representing;

(b4) nucleotide sequence of in SEQ ID NO:112, representing;

(b5) nucleotide sequence of in SEQ ID NO:154, representing;

(b6) nucleotide sequence of in SEQ ID NO:155, representing;

(b7) nucleotide sequence of in SEQ ID NO:156, representing;

(b8) nucleotide sequence of in SEQ ID NO:157, representing;

(b9) nucleotide sequence of in SEQ ID NO:158, representing;

(b10) nucleotide sequence of in SEQ ID NO:255, representing;

(b11) nucleotide sequence of in SEQ ID NO:257, representing;

(b12) nucleotide sequence of in SEQ ID NO:258, representing;

(b13) nucleotide sequence of in SEQ ID NO:259, representing;

(b14) nucleotide sequence of in SEQ ID NO:260, representing;

(b15) nucleotide sequence of in SEQ ID NO:261, representing;

(b16) nucleotide sequence of in SEQ ID NO:262, representing;

(b17) nucleotide sequence of in SEQ ID NO:263, representing;

(b18) nucleotide sequence of in SEQ ID NO:264, representing; With

(b19) a kind of nucleotide sequence, its with at SEQ ID NO:15, SEQ ID NO:16, SEQID NO:17, SEQ ID NO:112, SEQ ID NO:154, SEQ ID NO:155, SEQ IDNO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:255, SEQ IDNO:257, SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:260, SEQ IDNO:261, SEQ ID NO:262, the nucleotide sequence of any one expression has at least 90% sequence identity among SEQ ID NO:263 or the SEQ ID NO:264

29. carrier that comprises according to above-mentioned 28 DNA;

30. a transformant wherein will be introduced host cell according to above-mentioned 28 DNA;

31. a ferredoxin, it is selected from the group of being made up of following substances:

32. a DNA who comprises nucleotide sequence, described nucleotide sequence is selected from the group of being made up of following substances:

(ab1) nucleotide sequence of in SEQ ID NO:9, representing;

(ab2) nucleotide sequence of in SEQ ID NO:10, representing;

(ab3) nucleotide sequence of in SEQ ID NO:11, representing;

(ab4) nucleotide sequence of in SEQ ID NO:110, representing;

(ab5) nucleotide sequence of in SEQ ID NO:144, representing;

(ab6) nucleotide sequence of in SEQ ID NO:145, representing;

(ab7) nucleotide sequence of in SEQ ID NO:146, representing;

(ab8) nucleotide sequence of in SEQ ID NO:147, representing;

(ab9) nucleotide sequence of in SEQ ID NO:148, representing;

(ab10) nucleotide sequence of in SEQ ID NO:235, representing;

(abll) nucleotide sequence of in SEQ ID NO:236, representing;

(ab12) nucleotide sequence of in SEQ ID NO:237, representing;

(ab13) nucleotide sequence of in SEQ ID NO:238, representing;

(ab14) nucleotide sequence of in SEQ ID NO:239, representing;

(ab15) nucleotide sequence of in SEQ ID NO:240, representing;

(ab16) nucleotide sequence of in SEQ ID NO:241, representing;

(ab17) nucleotide sequence of in SEQ ID NO:242, representing;

(ab18) nucleotide sequence of in SEQ ID NO:243, representing; With

(ab19) nucleotide sequence of in SEQ ID NO:244, representing;

33. comprise carrier according to above-mentioned 32 DNA;

34. a transformant wherein will be introduced host cell according to above-mentioned 32 DNA;

35. the transformant according to above-mentioned 34, wherein said host cell are microorganism cells or vegetable cell;

36. a method of producing transformant, it comprises and will introduce the step of host cell according to above-mentioned 32 DNA;

37. a production has the method for protein that formula (II) compound is converted into the ability of formula (III) compound, described method comprises cultivation according to above-mentioned 34 transformant with reclaim the described proteinic step that produces;

38. a control method for weed, it comprises the step of compound administration in the proteic cultivation of plants of at least a herbicide metabolism of expression zone, and described herbicide metabolism albumen is selected from the group of being made up of following substances:

(A7) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise aminoacid sequence by dna encoding, described DNA is coded in SEQ ID NO:1 with comprising under rigorous condition, SEQ IDNO:2, the DNA of the nucleotide sequence of the aminoacid sequence of any one expression hybridization among SEQ ID NO:3 or the SEQ ID NO:108;

(A8) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise by the coded aminoacid sequence of DNA that can pass through polymerase chain reaction (PCR) amplification, primer that is included in the nucleotide sequence of representing among the SEQ ID NO:129 and the primer that is included in the nucleotide sequence of any one expression among the SEQ ID NO:124 to 128 are used in described polymerase chain reaction, and template is for belonging to the karyomit(e) that streptomyces (Streptomyces) or saccharopolyspora strain belong to (Saccharopo1yspora) microorganism;

(A9) be included in the protein of the aminoacid sequence of representing among the SEQ ID NO:4;

(A26) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise and at SEQ IDNO:159, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ IDNO:217, SEQ ID NO:219, SEQ ID NO:220, among SEQ ID NO:221 or the SEQ IDNO:223 aminoacid sequence of any one expression have at least 80% sequence identity aminoacid sequence or with at SEQ ID NO:160, SEQ ID NO:215, SEQ ID NO:216, SEQ IDNO:218, the aminoacid sequence that the aminoacid sequence of any one expression has at least 90% sequence identity among SEQ ID NO:222 or the SEQ ID NO:224; With

(A27) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise by nucleotide sequence coded aminoacid sequence, described nucleotide sequence be coded in SEQ ID NO:159, SEQ IDNO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ IDNO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ IDNO:219, SEQ ID NO:220, SEQ ID NO:221, SEQID NO:222, the nucleotide sequence of the aminoacid sequence of any one expression has at least 90% sequence identity among SEQ IDNO:223 or the SEQ ID NO:224

Wherein said compound is the compound of formula (I):

Wherein in formula (I), G is illustrated in the group of any one expression among the following G-1 to G-9:

Wherein in G-1 to G-9,

X represents Sauerstoffatom or sulphur atom;

Y represents Sauerstoffatom or sulphur atom;

R ¹Expression hydrogen atom or halogen atom;

R ²The expression hydrogen atom, C ₁-C ₈Alkyl, C ₁-C ₈Haloalkyl, halogen atom, hydroxyl ,-OR ⁹Group ,-SH group ,-S (O) pR ⁹Group ,-COR ⁹Group ,-CO ₂R ⁹Group ,-C (O) SR ⁹Group ,-C (O) NR ¹¹R ¹²Group ,-CONH ₂Group ,-CHO group ,-CR ⁹=NOR ¹⁸Group ,-CH=CR ¹⁹CO ₂R ⁹Group ,-CH ₂CHR ¹⁹CO ₂R ⁹Group ,-CO ²N=CR ¹³R ¹⁴Group, nitro, cyano group ,-NHSO ₂R ¹⁵Group ,-NHSO ₂NHR ¹⁵Group ,-NR ⁹R ²⁰Group ,-NH ₂Group or phenyl, they can be by one or more identical or different C ₁-C ₄Alkyl replaces;

P represents 0,1 or 2;

R ³Expression C ₁-C ₂Alkyl, C ₁-C ₂Haloalkyl ,-OCH ₃Group ,-SCH ₃Group ,-OCHF ₂Group, halogen atom, cyano group, nitro or C ₁-C ₃Alkoxyl group, it is used on the phenyl ring can be selected from halogen atom, C ₁-C ₃Alkyl, C ₁-C ₃Haloalkyl, OR ²⁸Group, NR ¹¹R ²⁸Group, SR ²⁸Group, cyano group, CO ₂R ²⁸The phenyl that at least one substituting group in group and the nitro replaces replaces;

R ⁴The expression hydrogen atom, C ₁-C ₃Alkyl, C ₁-C ₃Haloalkyl;

R ⁵The expression hydrogen atom, C ₁-C ₃Alkyl, C ₁-C ₃Haloalkyl, cyclopropyl, vinyl, C ₂Alkynyl, cyano group ,-C (O) R ²⁰Group ,-CO ₂R ²⁰Group ,-C (O) NR ²⁰R ²¹Group ,-CHR ¹⁶R ¹⁷The CN group ,-CR ¹⁶R ¹⁷C (O) R ²⁰Group ,-CR ¹⁶R ¹⁷CO ₂R ²⁰Group ,-CR ¹⁶R ¹⁷C (O) NR ²⁰R ²¹Group ,-CHR ¹⁶The OH group ,-CHR ¹⁶OC (O) R ²⁰Group or-OCHR ¹⁶OC (O) NR ²⁰R ²¹Group, or when G represents G-2 or G-6, R ⁴And R ⁵Can represent C=O group with they bonded carbon atoms;

R ⁶Expression C ₁-C ₆Alkyl, C ₁-C ₆Haloalkyl, C ₂-C ₆Alkoxyalkyl, C ₃-C ₆Thiazolinyl or C ₃-C ₆Alkynyl;

R ⁷The expression hydrogen atom, C ₁-C ₆Alkyl, C ₁-C ₆Haloalkyl, halogen atom ,-S (O) ₂(C ₁-C ₆Alkyl) or-C (=O) R ²²Group;

R ⁸The expression hydrogen atom, C ₁-C ₈Alkyl, C ₃-C ₈Cycloalkyl, C ₃-C ₈Thiazolinyl, C ₃-C ₈Alkynyl, C ₁-C ₈Haloalkyl, C ₂-C ₈Alkoxyalkyl, C ₃-C ₈Alkoxy alkoxy alkyl, C ₃-C ₈The halo alkynyl, C ₃-C ₈Haloalkenyl group, C ₁-C ₈Alkyl sulphonyl, C ₁-C ₈Halogenated alkyl sulfonyl, C ₃-C ₈Alkoxycarbonyl alkyl ,-S (O) ₂NH (C ₁-C ₈Alkyl) group ,-C (O) R ²³Group or can be by R on phenyl ring ²⁴The benzyl that replaces;

R ⁹Expression C ₁-C ₈Alkyl, C ₃-C ₈Cycloalkyl, C ₃-C ₈Thiazolinyl, C ₃-C ₈Alkynyl, C ₁-C ₈Haloalkyl, C ₂-C ₈Alkoxyalkyl, C ₂-C ₈Alkyl-thio-alkyl, C ₂-C ₈The alkyl sulphinyl alkyl, C ₂-C ₈The alkyl sulphonyl alkyl, C ₄-C ₈Alkoxy alkoxy alkyl, C ₄-C ₈Cycloalkylalkyl, C ₄-C ₈The cycloalkyloxy alkyl, C ₄-C ₈Alkene oxygen base alkyl, C ₄-C ₈The alkynyloxy group alkyl, C ₃-C ₈Halogenated alkoxy alkyl, C ₄-C ₈Haloalkene oxygen base alkyl, C ₄-C ₈Halo alkynyloxy group alkyl, C ₄-C ₈The cycloalkyl alkylthio, C ₄-C ₈The thiazolinyl alkylthio, C ₄-C ₈The alkynyl alkylthio, being used in can be by at least a halogen atom that is selected from, C on the ring ₁-C ₃Alkyl and C ₁-C ₃The C that the phenoxy group that the substituting group of haloalkyl replaces replaces ₁-C ₄Alkyl, being used in can be by at least a halogen atom that is selected from, C on the ring ₁-C ₃Alkyl and C ₁-C ₃The C that the benzyloxy that the substituting group of haloalkyl replaces replaces ₁-C ₄Alkyl, C ₄-C ₈The trialkylsyrylalkyl group, C ₂-C ₈Qing Wanji, C ₃-C ₈Halogenated cycloalkyl, C ₃-C ₈Haloalkenyl group, C ₅-C ₈The alkoxyl group thiazolinyl, C ₅-C ₈The halogenated alkoxy thiazolinyl, C ₅-C ₈The alkylthio thiazolinyl, C ₃-C ₈The halo alkynyl, C ₅-C ₈The alkoxyl group alkynyl, C ₅-C ₈The halogenated alkoxy alkynyl, C ₅-C ₈The alkylthio alkynyl, C ₂-C ₈Alkyl-carbonyl can be with at least a halogen atom that is selected from, C on ring ₁-C ₃Alkyl, C ₁-C ₃Haloalkyl ,-OR ²⁸Group ,-NR ¹¹R ²⁸Group ,-SR ²⁸Group, cyano group ,-CO ₂R ²⁸The benzyl that the substituting group of group and nitro replaces ,-CR ¹⁶R ¹⁷COR ¹⁰Group ,-CR ¹⁶R ¹⁷CO ₂R ²⁰Group ,-CR ¹⁶R ¹⁷P (O) (OR ¹⁰) ₂Group ,-CR ¹⁶R ¹⁷P (S) (OR ¹⁰) ₂Group ,-CR ¹⁶R ¹⁷C (O) NR ¹¹R ¹²Group ,-CR ¹⁶R ¹⁷C (O) NH ₂Group ,-C (=CR ²⁶R ²⁷) COR ¹⁰Group ,-C (=CR ²⁶R ²⁷) CO ₂R ²⁰Group ,-C (=CR ²⁶R ²⁷) P (O) (OR ¹⁰) ₂Group ,-C (=CR ²⁶R ²⁷) P (S) (OR ¹⁰) ₂Group ,-C (=CR ²⁶R ²⁷) C (O) NR ¹¹R ¹²Group ,-C (=CR ²⁶R ²⁷) C (O) NH ₂Group, or in the ring that Q-1 to Q-7 represents any one:

It can be by at least a halogen atom that is selected from, C on ring ₁-C ₆Alkyl, C ₁-C ₆Haloalkyl, C ₂-C ₆Thiazolinyl, C ₂-C ₆Haloalkenyl group, C ₂-C ₆Alkynyl, C ₃-C ₆The halo alkynyl, C ₂-C ₈Alkoxyalkyl ,-OR ²⁸Group ,-SR ²⁸Group ,-NR ¹¹R ²⁸Group, C ₃-C ₈Alkoxycarbonyl alkyl, C ₂-C ₄Carboxyalkyl ,-CO ₂R ²⁸The substituting group of group and cyano group replaces;

R ¹⁰Expression C ₁-C ₆Alkyl, C ₂-C ₆Thiazolinyl, C ₃-C ₆Alkynyl or tetrahydrofuran (THF) group;

R ¹¹And R ¹³Represent hydrogen atom or C independently ₁-C ₄Alkyl;

R ¹²Expression C ₁-C ₆Alkyl, C ₃-C ₆Cycloalkyl, C ₃-C ₆Thiazolinyl, C ₃-C ₆Alkynyl, C ₂-C ₆Alkoxyalkyl, C ₁-C ₆Haloalkyl, C ₃-C ₆Haloalkenyl group, C ₃-C ₆The halo alkynyl, the phenyl that on ring, can be replaced by at least a substituting group, described substituting group is selected from halogen atom, C ₁-C ₄Alkyl and C ₁-C ₄Alkoxyl group, or-CR ¹⁶R ¹⁷CO ₂R ²⁵Group; Or,

R ¹¹And R ¹²Can represent together-(CH ₂) ₅-,-(CH ₂) ₄-, or-CH ₂CH ₂OCH ₂CH ₂-, perhaps in that case, the ring of generation can be with being selected from C ₁-C ₃Alkyl, the substituting group of phenyl and benzyl replaces;

R ¹⁴Expression C ₁-C ₄Alkyl or the ring on can be with being selected from halogen atom, C ₁-C ₃Alkyl and C ₁-C ₃The phenyl that haloalkyl replaces; Or,

R ¹³And R ¹⁴Can represent C with they bonded carbon atoms ₃-C ₈Cycloalkyl;

R ¹⁵Expression C ₁-C ₄Alkyl, C ₁-C ₄Haloalkyl or C ₃-C ₆Thiazolinyl;

R ¹⁶And R ¹⁷Represent hydrogen atom or C independently ₁-C ₄Alkyl, C ₁-C ₄Haloalkyl, C ₂-C ₄Thiazolinyl, C ₂-C ₄Haloalkenyl group, C ₂-C ₄Alkynyl, C ₃-C ₄The halo alkynyl; Or,

R ¹⁶And R ¹⁷Can represent C with they bonded carbon atoms ₃-C ₆Cycloalkyl, perhaps the ring that so forms can be with at least a halogen atom that is selected from, C ₁-C ₃Alkyl and C ₁-C ₃The substituting group of haloalkyl replaces;

R ¹⁸The expression hydrogen atom, C ₁-C ₆Alkyl, C ₃-C ₆Thiazolinyl or C ₃-C ₆Alkynyl;

R ¹⁹The expression hydrogen atom, C ₁-C ₄Alkyl or halogen atom;

R ²⁰The expression hydrogen atom, C ₁-C ₆Alkyl, C ₃-C ₆Cycloalkyl, C ₃-C ₆Thiazolinyl, C ₃-C ₆Alkynyl, C ₂-C ₆Alkoxyalkyl, C ₁-C ₆Haloalkyl, C ₃-C ₆Haloalkenyl group, C ₃-C ₆The halo alkynyl can be with the phenyl of at least a substituting group replacement on ring, and described substituting group is selected from halogen atom, C ₁-C ₄Alkyl and-OR ²⁸Group, or-CR ¹⁶R ¹⁷CO ₂R ²⁵Group;

R ²¹The expression hydrogen atom, C ₁-C ₂Alkyl or-CO ₂(C ₁-C ₄Alkyl) group;

R ²²The expression hydrogen atom, C ₁-C ₆Alkyl, C ₁-C ₆Alkoxyl group or NH (C ₁-C ₆Alkyl) group;

R ²³Expression C ₁-C ₆Alkyl, C ₁-C ₆Haloalkyl, C ₁-C ₆Alkoxyl group, NH (C ₁-C ₆Alkyl) group, benzyl, C ₂-C ₈Dialkyl amido maybe can be used R ²⁴The phenyl that replaces;

R ²⁴Expression C ₁-C ₆Alkyl, 1-2 halogen atom, C ₁-C ₆Alkoxyl group or CF ₃Group;

R ²⁵Expression C ₁-C ₆Alkyl, C ₁-C ₆Haloalkyl, C ₃-C ₆Thiazolinyl, C ₃-C ₆Haloalkenyl group, C ₃-C ₆Alkynyl or C ₃-C ₆The halo alkynyl;

R ²⁶And R ²⁷Represent hydrogen atom independently of one another, C ₁-C ₄Alkyl, C ₁-C ₄Haloalkyl, C ₂-C ₄Thiazolinyl, C ₂-C ₄Haloalkenyl group, C ₂-C ₄Alkynyl, C ₃-C ₄The halo alkynyl ,-OR ²⁸Group ,-NHR ²⁸Group, or-SR ²⁸Group; Or,

R ²⁶And R ²⁷Can represent C with they bonded carbon atoms ₃-C ₈Cycloalkyl, or each ring that so forms can be with at least a halogen atom that is selected from, C ₁-C ₃Alkyl and C ₁-C ₃The substituting group of haloalkyl replaces; With

R ²⁸The expression hydrogen atom, C ₁-C ₆Alkyl, C ₁-C ₆Haloalkyl, C ₃-C ₆Thiazolinyl, C ₃-C ₆Haloalkenyl group, C ₃-C ₆Alkynyl, C ₃-C ₆The halo alkynyl, C ₂-C ₄Carboxyalkyl, C ₃-C ₈Alkoxycarbonyl alkyl, C ₃-C ₈The haloalkoxy carbonylic alkyl, C ₅-C ₉The allyloxycarbonyl alkyl, C ₅-C ₉Haloalkene oxygen base carbonylic alkyl, C ₅-C ₉The alkynyloxy group carbonylic alkyl, C ₅-C ₉Halo alkynyloxy group carbonylic alkyl, C ₅-C ₉Cyclo alkoxy carbonyl alkyl or C ₅-C ₉Halo cyclo alkoxy carbonyl alkyl;

39. a control method for weed, it comprises the step of compound administration at least a proteinic cultivation of plants of expression zone, and described protein is selected from the group of being made up of following substances:

40. a method of assessing cell to the resistance of formula (I) compound, described method comprises:

(1) with described compound and the step of expressing the proteic cells contacting of at least a herbicide metabolism, described herbicide metabolism albumen is selected from the group of being made up of the following formula material:

(A8) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise by the coded aminoacid sequence of DNA that can pass through polymerase chain reaction (PCR) amplification, primer that is included in the nucleotide sequence of representing among the SEQ ID NO:129 and the primer that is included in the nucleotide sequence of any one expression among the SEQ ID NO:124 to 128 are used in described polymerase chain reaction, and template is the chromosomal DNA that belongs to streptomyces or saccharopolyspora strain microorganism belonging to genus;

(2) be evaluated in the above-mentioned steps (1) step to the cells injury degree of contact compound;

41. the method according to above-mentioned 40, wherein said cell are microorganism cells or vegetable cell;

42. select the method for the cell of anti-formula (I) compound, described method comprises the resistance of assessing based in according to above-mentioned 40 method, selects the step of cell;

43. by the cell of the antiweed selected according to above-mentioned 42 method, or its culture;

44. a method of assessing plant to the resistance of formula (I) compound, described method comprises:

(1) with described compound and the step of expressing the proteic plant contact of at least a herbicide metabolism, described herbicide metabolism albumen is selected from the group of being made up of following substances:

(A8) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise by the coded aminoacid sequence of DNA that can pass through polymerase chain reaction (PCR) amplification, primer that is included in the nucleotide sequence of representing among the SEQ ID NO:129 and the primer that is included in the nucleotide sequence of any one expression among the SEQ ID NO:124 to 128 are used in described polymerase chain reaction, and template is the karyomit(e) that belongs to streptomyces or saccharopolyspora strain microorganism belonging to genus;

(2) assessment is to the step of the degree of injury of the plant of the contact compound of description in step (1);

45. a method of selecting the plant of anti-formula (I) compound, described method comprises the resistance of assessing based in according to above-mentioned 44 method, selects the step of plant;

46. one kind by the herbicide resistant plants of selecting according to above-mentioned 45 method, or its offspring;

47. the method for a processing formula (I) compound, described method is included under the existence of the electron transport system that contains electron donor described compound and at least a herbicide metabolism albumen test, and described herbicide metabolism albumen is selected from the group of being made up of following substances:

(A27) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise by nucleotide sequence coded aminoacid sequence, described nucleotide sequence be coded in SEQ ID NO:159, SEQ IDNO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ IDNO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ IDNO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, the nucleotide sequence of the aminoacid sequence of any one expression has at least 90% sequence identity among SEQ IDNO:223 or the SEQ ID NO:224;

48. method according to above-mentioned 47, wherein by described compound is contacted with transformant described compound and described herbicide metabolism albumen test, the proteic DNA of the described herbicide metabolism of will encoding in described transformant introduces the position that it is expressed in described cell;

49. the application of herbicide metabolism albumen in processing formula (I) compound, described herbicide metabolism albumen is selected from the group of being made up of following substances:

50. the application of the coding proteic polynucleotide of herbicide metabolism in processing formula (I) compound, described herbicide metabolism albumen is selected from the group of being made up of following substances

(A7) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise aminoacid sequence by dna encoding, described DNA is coded in SEQ ID NO:1 with comprising under rigorous condition, SEQ IDNO:2, the DNA hybridization of the nucleotide sequence of the aminoacid sequence of representing among SEQ ID NO:3 or the SEQ ID NO:108;

(A27) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise by nucleotide sequence coded aminoacid sequence, described nucleotide sequence be coded in SEQ ID NO:159, SEQ IDNO:160, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ IDNO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ IDNO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, the nucleotide sequence of the aminoacid sequence of any one expression has at least 90% sequence identity among SEQ IDNO:223 or the SEQ ID NO:224.

The accompanying drawing summary

Fig. 1 shows the annealing site of the PCR primer be used to obtain DNA of the present invention (A1) and DNA of the present invention (B1).Each numeral is meant SEQ ID number of expression primer nucleotide sequence.Arrow is represented to have with the annealing site of the Oligonucleolide primers of the nucleotide sequence of its SEQ ID number expression with from the bearing of trend of primed DNA polymeric enzyme reaction.Dotted line is represented the DNA by the pcr amplification that uses primer.Thick line represents that contiguous DNA inserts the carrier zone in site, and this carrier is used for producing chromosomal dna library.

Fig. 2 shows the annealing site of the PCR primer be used to obtain DNA of the present invention (A2) and DNA of the present invention (B2).Each numeral is meant SEQ ID number of expression primer nucleotide sequence.Arrow is represented to have with the annealing site of the Oligonucleolide primers of the nucleotide sequence of its SEQ ID number expression with from the bearing of trend of primed DNA polymeric enzyme reaction.Dotted line is represented the DNA by the pcr amplification that uses primer.Thick line represents that contiguous DNA inserts the carrier zone in site, and this carrier is used for producing chromosomal dna library.

Fig. 3 shows the annealing site of the PCR primer be used to obtain DNA of the present invention (A4) and DNA of the present invention (B4).Each numeral is meant SEQ ID number of expression primer nucleotide sequence.Arrow is represented to have with the annealing site of the Oligonucleolide primers of the nucleotide sequence of its SEQ ID number expression with from the bearing of trend of primed DNA polymeric enzyme reaction.Dotted line is represented the DNA by the pcr amplification that uses primer.Thick line represents that contiguous DNA inserts the carrier zone in site, and this carrier is used for producing chromosomal dna library.Yet, are the primers that insert the regional annealing in site with the contiguous DNA that is used for producing the carrier of chromosomal dna library by the Oligonucleolide primers of 57 expressions, fail to anneal with DNA of the present invention (A4).

Fig. 4 shows the restriction map of plasmid pKSN2.

Fig. 5 shows the restriction map of plasmid pCRrSt12.

Fig. 6 shows the restriction map of plasmid pCR657ET.

Fig. 7 shows the restriction map of plasmid pCR657FET.

Fig. 8 shows the restriction map of plasmid pCR657Bs.

Fig. 9 shows the restriction map of plasmid pCR657FBs.

Figure 10 shows the restriction map of plasmid pUCrSt12.

Figure 11 shows the restriction map of plasmid pUCrSt657.

Figure 12 shows the restriction map of plasmid pUCrSt657F.

Figure 13 shows the restriction map of plasmid pUCCR16G6-p/t.

Figure 14 shows by the oligonucleotide that will be made up of the nucleotide sequence of representing in SEQ ID NO:89 with by the anneal structure of joint NotI-EcoRI of generation of the oligonucleotide that the nucleotide sequence of representing in SEQ ID NO:90 is formed.

Figure 15 shows the restriction map of plasmid pUCCR16G6-p/t Δ.

Figure 16 shows by the oligonucleotide that will be made up of the nucleotide sequence of representing in SEQ ID NO:91 with by the anneal structure of joint HindIII-NotI of generation of the oligonucleotide that the nucleotide sequence of representing in SEQ ID NO:92 is formed.

Figure 17 shows the restriction map of plasmid pNdG6-Δ T.

Figure 18 shows the restriction map of plasmid pSUM-NdG6-rSt657.

Figure 19 shows the restriction map of plasmid pSUM-NdG6-rSt657F.

Figure 20 shows the restriction map of plasmid pKFrSt12.

Figure 21 shows the restriction map of plasmid pKFrSt12-657.

Figure 22 shows the restriction map of plasmid pKFrSt12-657F.

Figure 23 shows the restriction map of plasmid pSUM-NdG6-rSt12-657.

Figure 24 shows the restriction map of plasmid pSUM-NdG6-rSt12-657F.

Figure 25 shows by the oligonucleotide that will be made up of the nucleotide sequence of representing in SEQ ID NO:98 with by the anneal structure of joint HindIII-NotI-EcoRI of generation of the oligonucleotide that the nucleotide sequence of representing in SEQ ID NO:99 is formed.

Figure 26 shows the restriction map of plasmid pBI121S.

Figure 27 shows the restriction map of plasmid pBI-NdG6-rSt-657.

Figure 28 shows the restriction map of plasmid pBI-NdG6-rSt-657F.

Figure 29 shows the restriction map of plasmid pBI-NdG6-rSt12-657.

Figure 30 shows the restriction map of plasmid pBI-NdG6-rSt12-657F.

Figure 31 shows the restriction map of plasmid pCR923Sp.

Figure 32 shows the restriction map of plasmid pNdG6-rSt12.

Figure 33 shows the restriction map of plasmid pSUM-NdG6-rSt-923.

Figure 34 shows the restriction map of plasmid pKFrSt12-923.

Figure 35 shows the restriction map of plasmid pSUM-NdG6-rSt12-923.

Figure 36 shows the restriction map of plasmid pBI-NdG6-rSt-923.

Figure 37 shows the restriction map of plasmid pBI-NdG6-rSt12-923.

Figure 38 shows the restriction map of plasmid pCR671ET.

Figure 39 shows the restriction map of plasmid pCR671Bs.

Figure 40 shows the restriction map of plasmid pUCrSt671.

Figure 41 shows the restriction map of plasmid pSUM-NdG6-rSt-671.

Figure 42 shows the restriction map of plasmid pKFrSt12-671.

Figure 43 shows the restriction map of plasmid pSUM-NdG6-rSt12-671.

Figure 44 shows the restriction map of plasmid pBI-NdG6-rSt-671.

Figure 45 shows the restriction map of plasmid pBI-NdG6-rSt12-671.

Figure 46 shows that this PCR uses the oligonucleotide of the partial nucleotide sequence with DNA of the present invention (A) as primer by detect the result by the DNA of pcr amplification with agarose gel

electrophoresis.Swimming lane

1,7, the electrophoresis of 8,12,19,26,27,32,37,42 and 47 expression dna markers (ф 174/HaeIII digestion).Other swimming lane is represented the electrophoresis of sample shown in table 20 and 21.

Figure 47 shows by the oligonucleotide that will be made up of the nucleotide sequence of representing in SEQ ID NO:134 with by the anneal structure of joint of generation of the oligonucleotide that the nucleotide sequence of representing in SEQ ID NO:135 is formed.

Figure 48 shows the restriction map of plasmid pUCrSt657soy.

Figure 49 shows the restriction map of plasmid pSUM-NdG6-rSt-657soy.

Figure 50 shows the restriction map of plasmid pKFrSt12-657soy.

Figure 51 shows the restriction map of plasmid pSUM-NdG6-rSt12-657soy.

Figure 52 shows the restriction map of plasmid pBI-NdG6-rSt-657soy.

Figure 53 shows the restriction map of plasmid pBI-NdG6-rSt12-657soy.

Figure 54 shows the restriction map of plasmid pUCrSt1584soy.

Figure 55 shows the restriction map of plasmid pSUM-NdG6-rSt-1584soy.

Figure 56 shows the restriction map of plasmid pKFrSt12-1584soy.

Figure 57 shows the restriction map of plasmid pSUM-NdG6-rSt12-1584soy.

Figure 58 shows the restriction map of plasmid pBI-NdG6-rSt-1584soy.

Figure 59 shows the restriction map of plasmid pBI-NdG6-rSt12-1584soy.

Figure 60 shows the restriction map of plasmid pUCrSt1609soy.

Figure 61 shows the restriction map of plasmid pSUM-NdG6-rSt-1609soy.

Figure 62 shows by the oligonucleotide that will be made up of the nucleotide sequence of representing in SEQ ID NO:402 with by the anneal structure of joint EcoT22I-12aa-EcoT22I of generation of the oligonucleotide that the nucleotide sequence of representing in SEQ ID NO:403 is formed.

Figure 63 shows the restriction map of plasmid pUCrSt12-1609soy.

Figure 64 shows the restriction map of plasmid pSUM-NdG6-rSt12-1609soy.

Figure 65 shows the restriction map of plasmid pBI-NdG6-rSt-1609soy.

Figure 66 shows the restriction map of plasmid pBI-NdG6-rSt12-1609soy.

Explained later is in the described abbreviation of last figure:

DNA A1: DNA of the present invention (A1)

DNA A2: DNA of the present invention (A2)

DNA A3: DNA of the present invention (A3)

DNA A4: DNA of the present invention (A4)

DNA B1: DNA of the present invention (B1)

DNA B2: DNA of the present invention (B2)

DNA B4: DNA of the present invention (B4)

DNA A1S: DNA of the present invention (A1) S

DNA A23S: DNA of the present invention (A23) S

DNA A25S: DNA of the present invention (A25) S

Tac p:tac promotor

RrnB t:rrnB terminator

ColE1 ori: the replication orgin of plasmid ColE1

Amp ^r: ampicillin resistance gene

RuBPCssCTT: coding soybean (cv.Jack) ribulose-1,5-bisphosphate, the nucleotide sequence of the chloroplast transit peptides of the small subunit of 5-bisphosphate carboxylase

12aa: proteic 12 the amino acid whose nucleotide sequences of encoding mature, it is at soybean (cv.Jack) ribulose-1,5-bisphosphate, after the chloroplast transit peptides of the small subunit of 5-bisphosphate carboxylase

Km ^r: kalamycin resistance gene

F1 ori: the replication orgin of plasmid F1

The CR16G6p:CR16G6 promotor

The CR16t:CR16 terminator

CR16t Δ: the DNA that the nucleotide sequence in the restriction site downstream of restriction enzyme ScaI is removed from the CR16 terminator wherein

CR16G6p Δ: the DNA that the nucleotide sequence of the restriction site upstream of restriction enzyme NdeI is removed from the CR16G6 terminator wherein

NOSp: the promotor of nopaline synthase gene

NPTII: kalamycin resistance gene

NOSt: the terminator of nopaline synthase gene

GUS: β-glucuronidase gene

The right hand edge sequence of RB:T-DNA

The left hand edge sequence of LB:T-DNA

NdeI, HindIII, BspHI, EcoRI, BamHI, EcoT221, SphI, KpnI, SacI, BglII, NotI, ScaI: the restriction site of various restriction enzymes

Implement best way of the present invention

Below explain in detail the present invention.

The herbicide metabolism albumen that is selected from following protein combination (hereinafter being sometimes referred to as protein of the present invention (A)) has the ability that formula (II) compound (hereinafter being sometimes referred to as " compound (II) ") is converted into formula (III) compound (hereinafter being sometimes referred to as " compound (III) ").

＜protein combination 〉

(A28) a kind of protein, it has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor, and comprise by the coded aminoacid sequence of DNA that can pass through polymerase chain reaction (PCR) amplification, primer that is included in the nucleotide sequence of any one expression among the SEQ ID NO:124 to 128 and the primer that is included in the nucleotide sequence of representing among the SEQ ID NO:129 are used in described polymerase chain reaction, and template is an abortion within the first month of pregnancy look streptomycete, brick-red streptomycete, do not produce the look streptomycete, the brown streptomycete of ash, hot lightskyblue streptomycete, black walnut streptomycete, Tianjin island chain mould, pelletizing produces the look streptomycete, olive produces look streptomycete, decorative chains mould, streptomyces griseus, the wool streptomycete, three damp streptomycetes, pale asphyxia streptomycete, the rose streptomycete that reddens, ground, Shandong streptomycete, the chromosomal DNA of Regensburg streptomycete or Ta Shi saccharopolyspora strain.

As the specific examples of protein of the present invention (A), mention:

Be included in the protein (hereinafter being sometimes referred to as " protein of the present invention (A1) ") of the aminoacid sequence of representing among the SEQ ID NO:1;

Be included in the protein (hereinafter being sometimes referred to as " protein of the present invention (A2) ") of the aminoacid sequence of representing among the SEQ ID NO:2;

Be included in the protein (hereinafter being sometimes referred to as " protein of the present invention (A3) ") of the aminoacid sequence of representing among the SEQ ID NO:3;

Be included in the protein (hereinafter being sometimes referred to as " protein of the present invention (A4) ") of the aminoacid sequence of representing among the SEQ ID NO:108;

Be included in the protein (hereinafter being sometimes referred to as " protein of the present invention (A11) ") of the aminoacid sequence of representing among the SEQ ID NO:159;

Be included in the protein (hereinafter being sometimes referred to as " protein of the present invention (A12) ") of the aminoacid sequence of representing among the SEQ ID NO:160;

Be included in the protein (hereinafter being sometimes referred to as " protein of the present invention (A13) ") of the aminoacid sequence of representing among the SEQ ID NO:136;

Be included in the protein (hereinafter being sometimes referred to as " protein of the present invention (A14) ") of the aminoacid sequence of representing among the SEQ ID NO:137;

Be included in the protein (hereinafter being sometimes referred to as " protein of the present invention (A15) ") of the aminoacid sequence of representing among the SEQ ID NO:138;

Be included in the protein (hereinafter being sometimes referred to as " protein of the present invention (A16) ") of the aminoacid sequence of representing among the SEQ ID NO:215;

Be included in the protein (hereinafter being sometimes referred to as " protein of the present invention (A17) ") of the aminoacid sequence of representing among the SEQ ID NO:216;

Be included in the protein (hereinafter being sometimes referred to as " protein of the present invention (A18) ") of the aminoacid sequence of representing among the SEQ ID NO:217;

Be included in the protein (hereinafter being sometimes referred to as " protein of the present invention (A19) ") of the aminoacid sequence of representing among the SEQ ID NO:218;

Be included in the protein (hereinafter being sometimes referred to as " protein of the present invention (A20) ") of the aminoacid sequence of representing among the SEQ ID NO:219;

Be included in the protein (hereinafter being sometimes referred to as " protein of the present invention (A21) ") of the aminoacid sequence of representing among the SEQ ID NO:220;

Be included in the protein (hereinafter being sometimes referred to as " protein of the present invention (A22) ") of the aminoacid sequence of representing among the SEQ ID NO:221;

Be included in the protein (hereinafter being sometimes referred to as " protein of the present invention (A23) ") of the aminoacid sequence of representing among the SEQ ID NO:222;

Be included in the protein (hereinafter being sometimes referred to as " protein of the present invention (A24) ") of the aminoacid sequence of representing among the SEQ ID NO:223; With

Be included in the protein (hereinafter being sometimes referred to as " protein of the present invention (A25) ") of the aminoacid sequence of representing among the SEQ ID NO:224;

For example, PPO inhibition type herbicidal compound (hereinafter being sometimes referred to as " compound (I) ") and protein of the present invention (A) reaction by with formula (I) can change into this compound the compound with low weeding activity.

In addition, in the processing of the compound that compound (I) is converted into low weeding activity, can also utilize the herbicide metabolism albumen (hereinafter being sometimes referred to as " protein of the present invention (A) ") that is selected from following combination:

As the example of protein of the present invention (A), can mention above-described a-protein of the present invention.In addition, as other embodiment, can mention protein (hereinafter being sometimes referred to as " protein of the present invention (A9) ") that is included in the aminoacid sequence of representing among the SEQ ID NO:4 and the protein (hereinafter being sometimes referred to as " protein of the present invention (A10) ") that is included in the aminoacid sequence of representing among the SEQID NO:5.

In the combination of above-mentioned protein at (A5), (A6), (A7), (A8), (A26), (A27) or (A28) in the proteinic aminoacid sequence of expression, from SEQ ID NO:1,2,3,108,159,160,136,137,138,215,216,217,218,219,220,221, can observed difference in the aminoacid sequence of expression in 222,223 or 224 be some amino acid whose disappearance for example, replace, and insert.These differences comprise for example from being included in SEQ ID NO:1,2,3,108,159,160,136,137,138,215,216,217, the disappearance of the processing that the above-mentioned protein of the aminoacid sequence of expression is accepted in cell in 218,219,220,221,222,223 or 224.In addition, comprise abiogenous polymorphic variation, it is by the species that for example obtain proteinic biology, and the difference of individuality etc. causes; By passing through for example site-directed mutagenesis method, random mutagenesis method, the aminoacid deletion that manually-injected genetic mutation such as mutagenic treatment causes, replacement, and insertion.

Stand these disappearances, the amino acid whose quantity of replacing and inserting can be in protein of the present invention (A) can develop the scope of the ability that compound (II) is changed into compound (III).In addition, as amino acid whose replacement, can mention for example replacing in hydrophobicity electric charge, pK, the amino acid that aspects such as Stereo structure Characteristics are similar.Replace as these, for example mention the replacement in following each group particularly: (1) glycine and L-Ala; (2) Xie Ansuan, Isoleucine and leucine; (3) aspartic acid, L-glutamic acid, l-asparagine and glutamine; (4) Serine and Threonine; (5) Methionin and arginine; (6) phenylalanine and tyrosine; Deng.

In addition, in protein of the present invention (A), be present in preferably that to aim at shown in the SEQ ID NO:1 halfcystine in the site of No. 357 amino acid whose halfcystines in the aminoacid sequence are (do not stand disappearance or replace) of guarding: the example of this halfcystine is included in shown in the SEQ ID NO:2 in the aminoacid sequence halfcystine shown in the amino acid No. 350, the halfcystine shown in No. 344 amino acid in aminoacid sequence shown in the SEQ ID NO:3, the halfcystine shown in No. 360 amino acid in aminoacid sequence shown in the SEQ ID NO:108, the halfcystine shown in No. 359 amino acid in aminoacid sequence shown in the SEQ ID NO:4, the halfcystine shown in No. 355 amino acid in aminoacid sequence shown in the SEQ ID NO:5, the halfcystine shown in No. 358 amino acid in aminoacid sequence shown in the SEQ ID NO:159, the halfcystine shown in No. 374 amino acid in aminoacid sequence shown in the SEQ ID NO:160, the halfcystine shown in No. 351 amino acid in aminoacid sequence shown in the SEQ ID NO:136, the halfcystine shown in No. 358 amino acid in aminoacid sequence shown in the SEQ ID NO:137, the halfcystine shown in No. 358 amino acid in aminoacid sequence shown in the SEQID NO:138, the halfcystine shown in No. 347 amino acid in aminoacid sequence shown in the SEQ IDNO:222, halfcystine shown in No. 347 amino acid etc. in aminoacid sequence shown in the SEQ IDNO:224.

As manually causing these aminoacid deletion, insert or replace the method for (hereinafter being called amino acid modified sometimes jointly), for example mention being included in being coded in SEQ ID NO:1,2,3,108,159,160,136,137,138,215,216,217,218,219,220,221, carry out site-directed mutagenesis in 222,223 or 224 on the DNA of the aminoacid sequence of any one expression and make the method for the step that this DNA expresses then by ordinary method.As site-directed mutagenesis method, for example mention amber mutation (gapped duplex method, Nucleic Acids Res., 12,9441-9456 (the 1984)) method of using, the method for a kind of PCR introducing sudden change of passing through the use primer etc.In addition, as manually modified amino acid whose method, for example mention being included in being coded in SEQ ID NO:1,2,3,108,159,160,136,137,138,215,216,217,218,219,220,221, carry out random mutagenesis on the DNA of any one aminoacid sequence of expression in 222,223 or 224, make the method for the step of this DNA expression then by ordinary method.As the random mutagenesis method, for example mention by any one DNA in the above-mentioned aminoacid sequence of will encoding as template and right by the primer of using each full length DNA that to increase, at dATP as substrate, dTTP, the concentration of each is different under the common condition or promotes the Mg of polymeric enzyme reaction therein among dGTP and the dCTP ²⁺Concentration is increased to and is higher than the method for carrying out PCR under the common condition.As these PCR method, for example mention Biology, (31), 1994, the method for describing among the 97-112 at Method in Molecular.In addition, can mention the method for in PCT patent application publication number WO00/09682, describing.

In the present invention, " sequence identity " is meant homology and the identity between two nucleotide sequences or two aminoacid sequences.Should " sequence identity " can be by two sequences of region-wide comparison at cycle tests, each with optimum state to recently determining.Equally, insert or lack the optimum contrast that (for example breach) can be used for test oligonucleotide sequence or aminoacid sequence.This sequence identity can be by service routine such as FASTA (Pearson ﹠amp; Lipman, Proc.Natl.Acad.Sci.USA, 4,2444-2448 (1988)), BLAST (Altschul etc., Journal of Molecular Biology, 215,403-410 (1990)), CLUSTAL W (Thompson, Higgins ﹠amp; Gibson, Nucleic AcidResearch, 22,4673-4680 (1994a)) etc. the homology analysis correlated step of generation of carrying out calculate.These programs for example can typically go up at the webpage (http://www.ddbj.nig.ac.jp) of Japanese DNA database (at the international data center of Japanimation biology and the operation of DNA database hub) and utilize.In addition, sequence identity can be determined by using commercially available sequence analysis software.Particularly for example, (Software DevelopmentCompany is Ltd.) by Lipman-Pearson method (Lipman can to use GENETYX-WIN the 5th edition, DJ. and Pearson, W.R., Science, 227,1435-1441, (1985)) homology analysis produce recently calculating it.

About (A7) described " rigorous condition ", for example can mention according to as Sambrook, J., Frisch, E.F., and Maniatis, T., Molecular Cloning second edition, hybrid forms and uses then 2xSSC (Molecular Biology, John Wiley﹠amp in the hybridization that the described ordinary method of Cold SpringHarbor Press is carried out under 50 ℃ in the solution that is comprising 6xSSC (making the solution that comprises 1.5M NaCl and 0.15M trisodium citrate is 10xSSC) under 45 ℃; Sons, N.Y. (1989), 6.3.1-6.3.6) condition of washing hybrid.Can be for example from the condition of 2xSSC (low rigorous condition) to the condition (high rigorous condition) of 0.2xSSC salt concn in the selection washing step.Can for example from room temperature (low rigorous condition) to 65 ℃ (high rigorous conditions), select the temperature in the washing step.Alternatively, salt concn and temperature can change.

DNA as " being coded in SEQ ID NO:1 with comprising under rigorous condition, SEQ ID NO:2, the DNA of the amino acid whose nucleotide sequence of any one expression hybridization among SEQID NO:3 or the SEQ ID NO:108 " for example can mention comprising being coded in SEQ ID NO:1 particularly, 2,3,4,5,108,159,160,136,137,138,215,216,217,218,219,220, the DNA of the nucleotide sequence of the aminoacid sequence of any one expression in 221,222,223 or 224 is included in SEQ ID NO:6,7,8,78,84,109,139,140,141,142,143,225,226,227,228,229,230, DNA of the nucleotide sequence of any one expression etc. in 231,232,233 or 234.Also can mention comprise with in SEQ ID NO:6,7,8,78,84,109,139,140,141,142,143,225,226,227, the nucleotide sequence of any one expression has the DNA at least about the nucleotide sequence of 60% identity in 228,229,230,231,232,233 or 234.

As the molecular weight of identifying by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (hereinafter referred to as " SDS-PAGE "), the molecular weight of protein of the present invention (A) is about 30,000-60,000, typically about 40,000-50,000 (for example is comparable to by in SEQ ID NO:1,2,3,108,159,160,136,137,138,215,216,217,218,219, the protein that the aminoacid sequence of any one expression is formed in 220,221,222,223 or 224).In addition, as long as the ability that compound (II) is converted into compound (II) do not eliminate, protein of the present invention (A) can be as the protein that adds aminoacid sequence to its N-terminal upstream or its C-terminal downstream.

As the sign of the ability of the PPO inhibition type herbicidal compound of protein of the present invention (A) metabolism formula (I), can mention the ability that formula (II) compound is converted into formula (III) compound.This ability for example can be by confirming compound (II) in the presence of the electron transport system that comprises electron donor such as coenzyme NADP 11 with protein of the present invention (A) reaction with by detecting the compound (III) that produces.

" electron transport system that comprises electron donor " is meant that wherein redox chain reaction generation and electronics are transferred to the system of protein of the present invention (A) from electron donor.As electron donor, for example mention coenzyme NADP 11, NADH etc.For example, as the protein that can constitute electron transport system, mention ferredoxin and ferredoxin-NADP from NADPH to protein of the present invention (A) ⁺Reductase enzyme, the NADPH-cytochrome P-450 reductase, etc.

In order to confirm compound (II) is converted into the ability of compound (III), for example will comprise protein of the present invention (A), β-NADPH, ferredoxin, ferredoxin-NADP ⁺Reductase enzyme and with the reaction soln of about pH7 of the compound (II) of labelled with radioisotope about 10 minutes to 1 hour at about 30 ℃ of following incubations.Subsequently, after making reaction soln become acidity, use ethyl acetate extraction by adding hydrochloric acid.After the ethyl acetate layer that will reclaim carries out thin-layer chromatography (hereinafter referred to as " TLC "), carry out radioautograph, can confirm compound (II) is converted into the ability of compound (III) by the compound (III) of certification mark.

In order to prepare protein of the present invention (A), for example at first, according to conventional genetic engineering method (for example, at Sambrook, J., Frisch, E.F., Maniatis, T.; Molecular Cloning second edition, the method for describing among the Cold Spring Harbor Laboratory press) obtains the DNA (hereinafter being called " DNA of the present invention (A) " sometimes jointly) of code book invention protein (A).

As the example of DNA of the present invention (A), can mention the DNA (below be sometimes referred to as " DNA of the present invention (A) ") of code book invention protein (A).As the specific examples of DNA of the present invention (A), can mention:

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (A1) ") of aminoacid sequence shown in the SEQ ID NO:1;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (A2) ") of aminoacid sequence shown in the SEQ ID NO:2;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (A3) ") of aminoacid sequence shown in the SEQ ID NO:3;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (A4) ") of aminoacid sequence shown in the SEQ ID NO:108;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (A11) ") of aminoacid sequence shown in the SEQ ID NO:159;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (A12) ") of aminoacid sequence shown in the SEQ ID NO:160;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (A13) ") of aminoacid sequence shown in the SEQ ID NO:136;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (A14) ") of aminoacid sequence shown in the SEQ ID NO:137;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (A15) ") of aminoacid sequence shown in the SEQ ID NO:138;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (A16) ") of aminoacid sequence shown in the SEQ ID NO:215;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (A17) ") of aminoacid sequence shown in the SEQ ID NO:216;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (A18) ") of aminoacid sequence shown in the SEQ ID NO:217;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (A19) ") of aminoacid sequence shown in the SEQ ID NO:218;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (A20) ") of aminoacid sequence shown in the SEQ ID NO:219;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (A21) ") of aminoacid sequence shown in the SEQ ID NO:220;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (A22) ") of aminoacid sequence shown in the SEQ ID NO:221;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (A23) ") of aminoacid sequence shown in the SEQ ID NO:222;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (A24) ") of aminoacid sequence shown in the SEQ ID NO:223;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (A25) ") of aminoacid sequence shown in the SEQ ID NO:224; Deng.

In addition, as the example more specifically of DNA of the present invention (A), can mention:

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:6;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:9;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:7;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:10;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:8;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:11;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:109;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:110;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:139;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:144;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:140;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:145;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:141;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:146;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:142;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:147;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:143;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:148;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:225;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:235;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:226;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:236;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:227;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:237;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:228;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:238;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:229;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:239;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:230;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:240;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:231;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:241;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:232;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:242;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:233;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:243;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:234;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:244;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:214;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:368;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:393;

The coding have the ability that in the presence of the electron transport system that comprises electron donor, formula (II) compound is converted into formula (III) compound protein and with at SEQ ID NO:6, the nucleotide sequence of any one expression has the DNA of at least 80% sequence identity in 7,8 or 109;

The coding have the ability that in the presence of the electron transport system that comprises electron donor, formula (II) compound is converted into formula (III) compound protein and with in SEQ ID NO:139,140,141,142,143,225,226,227,228,229,230, the nucleotide sequence of any one expression has the DNA of at least 90% sequence identity in 231,232,233 or 234; Deng.

In addition, as the example of DNA of the present invention (A), except above DNA of the present invention (A), mention:

The DNA that comprises the nucleotide sequence of coded protein, described protein are included in the aminoacid sequence of representing among the SEQ ID NO:4 (hereinafter being sometimes referred to as " DNA of the present invention (A9) ");

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:78;

The DNA that comprises the nucleotide sequence of coded protein, described protein are included in the aminoacid sequence of representing among the SEQ ID NO:5 (hereinafter being sometimes referred to as " DNA of the present invention (A10) ");

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:84;

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:85; Deng.

DNA of the present invention (A) for example, can be from occurring in nature clone's DNA or can be by as the site-directed mutagenesis method, the random mutagenesis method with the disappearance of Nucleotide, introduce from the DNA of occurring in nature clone's DNA and can be the DNA of synthetic by replacement or insertion.Subsequently, protein of the present invention (A) can be produced by the DNA of the present invention (A) that expresses acquisition according to conventional genetic engineering method or obtain.Like this, can prepare protein of the present invention (A).

For example prepare DNA of the present invention (A) by the following method.At first, by conventional genetic engineering method as at Molecular Cloning:A Laboratory Manual second edition (1989), ColdSpring Harbor Laboratory Press; With Current Protocols in MolecularBiology (1987), John Wiley ﹠amp; Those that describe in the Sons company, from microorganism, prepare chromosomal DNA, described microorganism belongs to streptomyces, as abortion within the first month of pregnancy look streptomycete, brick-red streptomycete, do not produce the look streptomycete, light gray streptomycete (Streptomyces griseolus), Streptomycescarbophilus, the brown streptomycete of ash, hot lightskyblue streptomycete, the black walnut streptomycete, Tianjin island chain mould, pelletizing produces the look streptomycete, and olive produces the look streptomycete, the decorative chains mould, streptomyces griseus, wool streptomycete, three damp streptomycetes, the pale asphyxia streptomycete, the rose streptomycete that reddens, Shandong ground streptomycete and Regensburg streptomycete, more specifically, abortion within the first month of pregnancy look streptomycete IFO 12898, brick-red streptomycete ATCC 21469 does not produce look streptomycete IFO 12735, light gray streptomycete ATCC 11796, Streptomycescarbophilus SANK62585, the brown streptomycete IFO 12870t of ash, hot lightskyblue streptomycete IFO14273t, black walnut streptomycete IFO 13445, Tianjin island chain mould IFO 13782, pelletizing produces look streptomycete IFO 13673t, and olive produces look streptomycete IFO 12444, decorative chains mould IFO 13069t, streptomyces griseus ATCC 10137, streptomyces griseus IFO 13849T, wool streptomycete IFO 12787T, three damp streptomycete IFO 13855T, pale asphyxia streptomycete IFO 13434T, the rose streptomycete IFO13682T that reddens, Shandong ground streptomycete IFO 15875T and Regensburg streptomycete IFO 13446T, etc.; Or belong to the microorganism that saccharopolyspora strain belongs to, as the Ta Shi saccharopolyspora strain, more specifically, Ta Shi saccharopolyspora strain JCM9383t etc.Next, after partly digesting chromosomal DNA, reclaim the DNA of about 2kb with restriction enzyme such as Sau3AI.According in " Molecular Cloning:A LaboratoryManual second edition " (1989), Cold Spring Harbor Laboratory Press; " Current Protocolsin Molecular Biology " (1987), John Wiley ﹠amp; The conventional genetic engineering method of describing in the Sons company with the dna clone that reclaims in carrier.As carrier, particularly for example, can utilize pUC 119 (TaKaRa Shuzo Company), pTVA 118N (Takara Shuzo Company), pBluescript II (Toyobo Company), pCR2.1-TOPO (Invitrogen), pTrc99A (Amersham Pharmacia Biotech Company), pKK331-1A (AmershamPharmacia Biotech Company), etc.Can obtain chromosomal dna library by from the clone who obtains, extracting plasmid.

DNA of the present invention (A) can be by obtaining with the hybridization of the chromosomal dna library of probe and acquisition with by detection and recovery and probe specificity bonded DNA under the following conditions.Probe can be by about at least 20 DNA that Nucleotide is formed, and described Nucleotide comprises and is coded in SEQ IDNO:1, the nucleotide sequence of the aminoacid sequence of any one expression in 2,3 or 108.As can mentioning being included in SEQ ID NO:6 as the specific examples of the DNA of probe, the DNA of the nucleic acid of any one expression in 7,8 or 109; Be included in SEQ ID NO:6, the DNA of the partial nucleotide sequence of the nucleotide sequence of any one expression in 7,8 or 109; Comprise DNA with described partial nucleotide sequence complementary nucleotide sequence; Deng.

Use radio isotope, marks such as fluorescent dye are as the DNA of probe.In order to use labelled with radioisotope DNA, for example can use the random labelling test kit of Boehringer or Takara shuzo Company.In addition, can prepare usefulness by carrying out PCR ³²The DNA of P mark.The DNA that will be used for probe is as template.With (α- ³²P) the dCTP exchange typically is used in the dCTP in the PCR reaction soln.In addition, when with the fluorescent dye marker DNA, for example can use DIG-High PrimeDNA labeling and Detection Starter test kit II (Roche Company).

Next explanation prepares the specific examples of probe.For example, by being used as template from the chromosomal DNA or the chromosomal dna library of aforesaid abortion within the first month of pregnancy look streptomycete IFO 12898 preparations, the oligonucleotide of forming by the oligonucleotide that will be made up of nucleotide sequence shown in the SEQ ID NO:93 with by nucleotide sequence shown in the SEQ ID NO:94 is as primer, with by use as described in following example for example PCR DIG probe synthetic agent box (Roche Diagnostics GmbH) carry out PCR according to the handbook of attaching, can obtain to comprise the full length nucleotide sequence shown in the SEQ ID NO:6, DNA with the digoxigenin mark.Similarly, by will be from the chromosomal DNA of aforesaid abortion within the first month of pregnancy look streptomycete IFO 12898 preparations or chromosomal dna library as template, can obtain to be included in represent among the SEQ IDNO:6 from Nucleotide 57 to Nucleotide 730 nucleotide sequence, with the DNA of digoxigenin mark.As primer, use oligonucleotide of forming by the nucleotide sequence shown in the SEQ ID NO:130 and the oligonucleotide of forming by the nucleotide sequence shown in the SEQ ID NO:131 to carry out PCR.In addition, by being used as template from the chromosomal DNA or the chromosomal dna library of aforesaid Ta Shi saccharopolyspora strain JCM 9383t preparation, the DNA that can obtain to comprise the full length nucleotide sequence shown in the SEQ ID NO:7, uses the digoxigenin mark.As primer, use oligonucleotide of forming by the nucleotide sequence shown in the SEQ IDNO:61 and the oligonucleotide of forming by the nucleotide sequence shown in the SEQ ID NO:62 to carry out PCR.In addition, by being used as template from the chromosomal DNA or the chromosomal dna library of aforesaid brick-red streptomycete ATCC 21469 preparations, the DNA that can obtain to comprise the full length nucleotide sequence shown in the SEQ ID NO:8, uses the digoxigenin mark.As primer, use oligonucleotide of forming by the nucleotide sequence shown in the SEQ ID NO:70 and the oligonucleotide of forming by the nucleotide sequence shown in the SEQ ID NO:71 to carry out PCR.In addition, by will be from the chromosomal DNA of aforesaid brick-red streptomycete ATCC 21469 preparations or chromosomal dna library as template, can obtain to be included in represent among the SEQ ID NO:8 from Nucleotide 21 to Nucleotide 691 nucleotide sequence, with the DNA of digoxigenin mark.As primer, use oligonucleotide of forming by the nucleotide sequence shown in the SEQ ID NO:132 and the oligonucleotide of forming by the nucleotide sequence shown in the SEQ IDNO:133 to carry out PCR.

Make the method for probe and chromosomal dna library hybridization can comprise colony hybridization and plaque hybridization, can select to prepare the compatible proper method of type of used carrier with the library.When used library is to use plamid vector construction, carry out colony hybridization.Particularly, at first, obtain transformant by the DNA in library being introduced the microorganism that the carrier that wherein is used to make up the library can duplicate.The transformant that dilution obtains is coated on the agar plate and cultivation occurs until bacterium colony.When phage vector being used to make up the library, carry out plaque hybridization.Particularly, at first, infecting under the possible condition, the microorganism that the phage vector that wherein is used to produce the library can be duplicated mixes with the phage in library.Then mixture is further mixed with soft agar.Then this mixture is coated on the agar plate.Subsequently, culturing mixt occurs until plaque.

Next, in the situation of arbitrary above-mentioned hybridization, film is placed on the agar plate surface of wherein carrying out above-mentioned cultivation, the bacterium colony of transformant or the phage particle in the plaque are transferred on the film.Behind the alkaline purification film, carry out neutralizing treatment.To be fixed on the film from the DNA of transformant or phage particle wash-out then.More specifically, for example in the incident of plaque hybridization, by with nitrocellulose membrane or nylon membrane, Hybond-N for example particularly ⁺(Amersham Pharmacia Biotech Company) is placed on the agar plate and waits for 1 minute phage particle is absorbed on the film.Film is immersed in the basic solution (1.5M NaCl and 0.5N NaOH) about 3 minutes to be eluted on the film with the dissolving phage particle with phage DNA.Then film is immersed in the neutralization solution (1.5M NaCl and 0.5Mtris-HCl pH of buffer 7.5) about 5 minutes.The washing film is about 5 minutes in washing soln (0.3M NaCl, 30mM Trisodium Citrate, 0.2M tris-HCl pH of buffer 7.5), for example by phage DNA being fixed on the film in about 90 minutes at about 80 ℃ of following incubations in a vacuum.

By using the film of preparation like this, use above-mentioned DNA to hybridize as probe.For example can be according to " Molecular Cloning:A Laboratory Manual second edition (1989) ", the description among the ColdSpring Harbor Laboratory Press etc. is hybridized.

Although can provide all temps condition and reagent to hybridize, the film of preparation is as mentioned above soaked under 42 ℃ to 65 ℃ and remains on 50 μ l-200 μ l/1cm ²In the prehybridization solution of the ratio of film preparation 1 hour to 4 hours.Prehybridization solution for example can comprise 450mM to 900mM NaCl and 45mM to 90mM Trisodium Citrate, comprise the sodium lauryl sulphate that concentration is 0.1%-1.0% (hereinafter referred to as " SDS "), with comprise the non-specific DNA that concentration is the sex change of 0 μ g/ml-200 μ g/ml, and can comprise sometimes separately that concentration is the white protein of 0%-0.2%, phycol and polyvinylpyrrolidone.Subsequently, for example film is soaked under 42 ℃ to 65 ℃ and remain on 50 μ l-200 μ l/1cm ²In the hybridization solution of the ratio of film preparation 12 hours to 20 hours.Hybridization solution be for example prehybridization solution and the probe that obtains with above-mentioned preparation method (relative populations is 1.0 x 10 ⁴Cpm-2.0 x 10 ⁶Cpm/1cm ²Film) mixture, this prehybridization solution can comprise 450mM to 900mMNaCl and 45mM to 90mM Trisodium Citrate, comprise the SDS that concentration is 0.1%-1.0%, with comprise the non-specific DNA that concentration is the sex change of 0 μ g/ml-200 μ g/ml, and can comprise sometimes separately that concentration is the white protein of 0%-0.2%, phycol and polyvinylpyrrolidone.Subsequently, remove film and use and comprise 15mM-300mM NaCl, 42 ℃ of-65 ℃ of washingss of 1.5mM-30mM Trisodium Citrate and 0.1%-1.0%SDS carry out 5 minutes to 15 minutes about 2-4 time of washing.In addition, with 2 x SSC solution (300mMNaCl and 30mM Trisodium Citrate) gently after the rinsing, with the film drying.By film being carried out the position of radioautograph detection probes on film, identify on the film position with the DNA of used probe hybridization.Alternatively, prehybridization and hybridization can be used commercially available hybridization kit, are included in DIG-HighPrime DNA Labeling ﹠amp as use; Hybridization solution among the Detection Starter test kit II (Roche) carries out.After hybridization, for example, in comprising the 2 x SSC of 0.1%SDS, at room temperature washed film twice 5 minutes, then in comprising the 0.5 x SSC of 0.1%SDS, washed film twice 15 minutes down at 65 ℃.By handling the film of washing and detect on film position with the DNA of used probe hybridization with being included in detection liquid in the test kit again by the position of detecting probe on the film.

The clone of the position of the DNA that detects on identifying corresponding to film on the primary nutrient agar, it can be separated the clone who carries those DNA by picking.

Can be according to " Molecular Cloning:A Laboratory Manual second edition " (1989), Cold Spring Harbor Laboratory Press, " Current Protocols in MolecularBiology " (1987), John Wiley ﹠amp; Described conventional genetic engineering methods such as Sons Incorporated will be cloned in the carrier according to the DNA of the present invention (A) of above acquisition.As carrier, particularly for example, can use pUCA119 (Takara Shuzo Company), pTVA118N (TakaraShuzo Company), pBluescript II (Toyobo Company), pCR2.1-TOPO (Invitrogen Company), pTrc99A (Pharmacia Company), pKK331-1A (Pharmacia Company) etc.

In addition, can be by at F.Sanger according to the nucleotide sequence of the DNA of the present invention (A) of above-mentioned acquisition, S.Nicklen, A.R.Coulson, the two deoxidation cessation method described in Proceeding of National Academy of ScienceU.S.A. (1977) 74:5463-5467 are analyzed.In the specimen preparation of nucleotide sequence analysis, can use commercially available reagent, as the ABI PRISM dyestuff termination cycle sequencing easy reaction test kit of Perkin Elmer company.

Can also be prepared as follows DNA of the present invention (A).Can be by carrying out pcr amplification DNA of the present invention (A).PCR can use as mentioned above from the chromosomal DNA of microorganism preparation or chromosomal dna library as template, described microorganism belongs to streptomyces, as abortion within the first month of pregnancy look streptomycete, brick-red streptomycete, do not produce the look streptomycete, the light gray streptomycete, Streptomyces carbophilus, the brown streptomycete of ash, hot lightskyblue streptomycete, the black walnut streptomycete, Tianjin island chain mould, pelletizing produces the look streptomycete, olive produces look streptomycete, decorative chains mould, streptomyces griseus, the wool streptomycete, three damp streptomycetes, pale asphyxia streptomycete, the rose streptomycete that reddens, Shandong ground streptomycete and Regensburg streptomycete, more specifically, abortion within the first month of pregnancy look streptomycete IFO 12898, brick-red streptomycete ATCC 21469, do not produce look streptomycete IFO12735, light gray streptomycete ATCC 11796, Streptomyces carbophilus SANK62585, grey brown streptomycete IFO 12870t, hot lightskyblue streptomycete IFO 14273t, black walnut streptomycete IFO13445, Tianjin island chain mould IFO 13782, pelletizing produce look streptomycete IFO 13673t, olive produces look streptomycete IFO 12444, decorative chains mould IFO 13069t, streptomyces griseus ATCC 10137, streptomyces griseus IFO 13849T, wool streptomycete IFO 12787T, three damp streptomycete IFO 13855T, pale asphyxia streptomycete IFO 13434T, the rose streptomycete IFO 13682T that reddens, Shandong ground streptomycete IFO15875T and Regensburg streptomycete IFO 13446T, etc.; Or belong to the microorganism that saccharopolyspora strain belongs to, as the Ta Shi saccharopolyspora strain, more specifically, Ta Shi saccharopolyspora strain JCM 9383t etc.PCR also can use and comprise coding SEQ ID NO:1,2,3,4,5,108,159,160,136,137,138,215,216,217,218,219,220,221, in 222,223 or 224 shown in any one 5 ' of the nucleotide sequence of aminoacid sequence terminal at least about 20 Nucleotide oligonucleotide and comprise with the above-mentioned aminoacid sequence of encoding in any one nucleotide sequence neighbour advance the oligonucleotide at least about 20 Nucleotide complementary nucleotide sequences in 3 ' end or 3 ' terminal downstream.PCR can carry out under the following conditions.At the above-mentioned 5 ' end side that is used for the primer of PCR, can increase the restriction enzyme recognition sequence.

More specifically for example, by using from the chromosomal DNA of abortion within the first month of pregnancy look streptomycete IFO 12898 preparations or chromosomal dna library as template, carry out PCR with the oligonucleotide that comprises nucleotide sequence shown in the SEQ ID NO:52 as primer with the oligonucleotide that comprises nucleotide sequence shown in the SEQ ID NO:51 by use and can prepare the DNA that comprises the nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO:1, comprise the DNA of nucleotide sequence shown in the SEQ ID NO:6 etc.Alternatively, the oligonucleotide that comprises the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:51 and comprise nucleotide sequence shown in the SEQID NO:53 by use carries out PCR as primer and can increase and comprise the DNA of nucleotide sequence shown in the SEQ ID NO:9 (nucleotide sequence that comprises aminoacid sequence shown in the coding SEQ ID NO:1).

For example, by using from the chromosomal DNA of Ta Shi saccharopolyspora strain JCM 9383t preparation or chromosomal dna library as template, carry out PCR with the oligonucleotide that comprises nucleotide sequence shown in the SEQ ID NO:62 as primer with the oligonucleotide that comprises nucleotide sequence shown in the SEQ ID NO:61 by use and can prepare the DNA that comprises the nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO:2, comprise the DNA of nucleotide sequence shown in the SEQ ID NO:7 etc.Alternatively, the oligonucleotide that comprises the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:61 and comprise nucleotide sequence shown in the SEQ ID NO:63 by use carries out PCR as primer and can increase and comprise the DNA of nucleotide sequence shown in the SEQ IDNO:10 (nucleotide sequence that comprises aminoacid sequence shown in the coding SEQ ID NO:2).

For example, by using the chromosomal DNA never produce look streptomycete IFO 12735 preparations or chromosomal dna library as template, carry out PCR with the oligonucleotide that comprises nucleotide sequence shown in the SEQ ID NO:120 as primer with the oligonucleotide that comprises nucleotide sequence shown in the SEQ ID NO:119 by use and can prepare the DNA that comprises the nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO:108, comprise the DNA of nucleotide sequence shown in the SEQ ID NO:109 etc.Alternatively, the oligonucleotide that comprises the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:119 and comprise nucleotide sequence shown in the SEQ IDNO:121 by use carries out PCR as primer and can increase and comprise the DNA of nucleotide sequence shown in the SEQID NO:110 (nucleotide sequence that comprises aminoacid sequence shown in the coding SEQ ID NO:108).

For example, by using from the chromosomal DNA of black walnut streptomycete IFO 13445 preparations or chromosomal dna library as template with carry out PCR by the oligonucleotide that use comprises the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:165 and comprises nucleotide sequence shown in the SEQ ID NO:166 as primer and can prepare the DNA that comprises nucleotide sequence shown in the SEQ ID NO:144 (nucleotide sequence that comprises aminoacid sequence shown in the coding SEQ ID NO:159).

For example, by using from the chromosomal DNA of Tianjin island chain mould IFO 13782 preparations or chromosomal dna library as template with carry out PCR by the oligonucleotide that use comprises the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:171 and comprises nucleotide sequence shown in the SEQ ID NO:172 as primer and can prepare the DNA that comprises nucleotide sequence shown in the SEQ ID NO:145 (nucleotide sequence that comprises aminoacid sequence shown in the coding SEQ ID NO:160).

For example, by using from the chromosomal DNA of hot lightskyblue streptomycete IFO 14273t preparation or chromosomal dna library as template with carry out PCR by the oligonucleotide that use comprises the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:177 and comprises nucleotide sequence shown in the SEQ ID NO:178 as primer and can prepare the DNA that comprises nucleotide sequence shown in the SEQ ID NO:146 (nucleotide sequence that comprises aminoacid sequence shown in the coding SEQ ID NO:136).

For example, produce the chromosomal DNA of look streptomycete IFO 13673t preparation or chromosomal dna library as template with carry out PCR by the oligonucleotide that use comprises the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:183 and comprises nucleotide sequence shown in the SEQ ID NO:184 as primer and can prepare the DNA that comprises nucleotide sequence shown in the SEQ ID NO:147 (nucleotide sequence that comprises aminoacid sequence shown in the coding SEQ ID NO:137) by using from pelletizing.

For example, produce the chromosomal DNA of look streptomycete IFO 12444 preparations or chromosomal dna library as template with carry out PCR by the oligonucleotide that use comprises the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:184 and comprises nucleotide sequence shown in the SEQ ID NO:185 as primer and can prepare the DNA that comprises nucleotide sequence shown in the SEQ ID NO:148 (nucleotide sequence that comprises aminoacid sequence shown in the coding SEQ ID NO:138) by using from olive.

When the DNA library that will wherein chromosomal DNA be introduced carrier is used as template, for example, also can comprise and be selected from coding SEQ ID NO:1 by use, 2,3,4,5,108,159,160,136, the oligonucleotide of the nucleotide sequence of the nucleotide sequence of any one aminoacid sequence shown in 137 or 138 (for example comprising the oligonucleotide of the nucleotide sequence 5 ' end side of aminoacid sequence shown in the coding SEQ ID NO:1) at least about the nucleotide sequence of 20 Nucleotide and comprise with the nucleotide sequence complementary nucleotide sequence in the contiguous carrier DNA insertion site that is used to make up the library carry out the PCR DNA of the present invention (A) that increases at least about the oligonucleotide of 20 Nucleotide as primer.5 ' end side at the primer that is used for PCR as mentioned above can add the restriction enzyme recognition sequence.

About the condition of above-mentioned this PCR, particularly for example, can mention following condition: keep 97 ℃ 2 minutes, repeat 10 circulations then, circulation comprise keep 97 ℃ 15 seconds, then 65 ℃ 30 seconds, then 72 ℃ 2 minutes; Carry out 15 circulations then, circulation comprise keep 97 ℃ 15 seconds, then 68 ℃ of 30 seconds and then 72 ℃ 2 minutes (each circulation increases by 20 seconds successively); Remain on then 72 ℃ 7 minutes.PCR can use the reaction soln of 50 μ l, it comprises the 50ng chromosomal DNA, comprise 2 kinds of primers of above-mentioned paired 300nM separately, comprise 5.0 μ l dNTP mixtures (mixture of every kind of 2.0mM of 4 kinds of dNTP), comprise 5.0 μ l 10x ExpandHF damping fluids and (comprise MgCl ₂, Roche Molecular Biochemicals Company) and comprise 0.75 μ l Expand HiFi enzyme mixture (Roche Molecular Biochemicals Company).

Alternatively, can mention following condition: keep 97 ℃ 2 minutes, repeat 30 circulations then, circulation comprise 97 ℃ 15 seconds, then 60 ℃ 30 seconds, then 72 ℃ 90 seconds, keep then reaction soln 72 ℃ 4 minutes.PCR can use the reaction soln of 50 μ l, it comprises the 250ng chromosomal DNA, comprise 2 kinds of primers of above-mentioned paired 200nM separately, comprise 5.0 μ l dNTP mixtures (mixture of every kind of 2.5mM of 4 kinds of dNTP), 5.0 μ l 10x ExTaq damping fluids (comprise MgCl ₂, Takara Shuzo Company) and comprise 0.5 μ l ExTaq polysaccharase (Takara ShuzoCompany).

Alternatively, for example based at SEQ ID NO:6, the nucleotide sequence in the extra high zone of sequence identity in the nucleotide sequence can design and prepare the oligonucleotide as primer shown in 7,8 or 109.DNA of the present invention (A) can also carry out PCR as primer and chromosomal DNA or chromosomal dna library by the oligonucleotide that use to obtain and obtain.Can from microorganism, prepare chromosomal DNA or chromosomal dna library as mentioned above, described microorganism belongs to streptomyces, as abortion within the first month of pregnancy look streptomycete, brick-red streptomycete, do not produce the look streptomycete, the light gray streptomycete, Streptomycescarbophilus, the brown streptomycete of ash, hot lightskyblue streptomycete, black walnut streptomycete, Tianjin island chain mould, pelletizing produces the look streptomycete, olive produces look streptomycete, decorative chains mould, streptomyces griseus, the wool streptomycete, three damp streptomycetes, pale asphyxia streptomycete, the rose streptomycete that reddens, Shandong ground streptomycete and Regensburg streptomycete, more specifically, abortion within the first month of pregnancy look streptomycete IFO 12898, brick-red streptomycete ATCC 21469, do not produce look streptomycete IFO 12735, light gray streptomycete ATCC 11796, Streptomycescarbophilus SANK62585, grey brown streptomycete IFO 12870t, hot lightskyblue streptomycete IFO14273t, black walnut streptomycete IFO 13445, Tianjin island chain mould IFO 13782, pelletizing produce look streptomycete IFO 13673t, olive produces look streptomycete IFO 12444, decorative chains mould IFO 13069t, streptomyces griseus ATCC 10137, streptomyces griseus IFO 13849T, wool streptomycete IFO 12787T, three damp streptomycete IFO 13855T, pale asphyxia streptomycete IFO 13434T, the rose streptomycete IFO13682T that reddens, Shandong ground streptomycete IFO 15875T and Regensburg streptomycete IFO 13446T, etc.; Or belong to the microorganism that saccharopolyspora strain belongs to, as the Ta Shi saccharopolyspora strain, more specifically, Ta Shi saccharopolyspora strain JCM9383t etc.About " at SEQ ID NO:6, shown in 7,8 or 109 in the nucleotide sequence the extra high zone of sequence identity ", for example, mention corresponding to Nucleotide 290-315 in the nucleotide sequence shown in the SEQ ID NO:6 458-485, the zone in zone shown in each among 496-525 or the 1046-1073.About primer, for example, can mention comprising the primer of nucleotide sequence shown in any one among the SEQ IDNO:124-129 based on these nucleotide sequence zone design.

SEQ ID NO:124; Based on nucleotide sequence corresponding to the zone in zone shown in the above Nucleotide 290-315;

SEQ ID NO:125; Based on nucleotide sequence corresponding to the zone in zone shown in the above Nucleotide 458-485;

SEQ ID NO:126; Based on nucleotide sequence corresponding to the zone in zone shown in the above Nucleotide 458-485;

SEQ ID NO:127; Based on nucleotide sequence corresponding to the zone in zone shown in the above Nucleotide 496-525;

SEQ ID NO:128; Based on nucleotide sequence corresponding to the zone in zone shown in the above Nucleotide 496-525; With

SEQ ID NO:129; Based on nucleotide sequence corresponding to the zone in zone shown in the above Nucleotide 1046-1073.

For example, comprise the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:124 and comprise the DNA of the pairing of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:129 by use as the about 800bp of primer amplification.Comprise the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:125 and comprise the DNA of the pairing of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:129 by use as the about 600bp of primer amplification.Comprise the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:126 and comprise the DNA of the pairing of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:129 by use as the about 600bp of primer amplification.Comprise the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:127 and comprise the DNA of the pairing of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:129 by use as the about 580bp of primer amplification.In addition, comprise the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:128 and comprise the DNA of the pairing of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:129 by use as the about 580bp of primer amplification.

About the PCR condition, particularly for example, mention following condition: keep 95 ℃ 1 minute; Repeat 30 circulations, circulation comprise keep 94 ℃ 15 seconds, then 60 ℃ 30 seconds, then 72 ℃ 1 minute; Remain on then 72 ℃ 5 minutes.Can use the reaction soln of 25 μ l, it comprises the 10ng chromosomal DNA, comprise 2 kinds of primers 200nM separately, comprise 0.5 μ, 1 dNTP mixture (mixture of every kind of 10mM of 4 kinds of dNTP), comprise 5 μ l 5x GC genome PCR reaction buffers, it comprises 5 μ l 5M GC-Melt and comprises 0.5 μ l Advantage-GC genome polysaccharase mixture (Clontech Company).

By reclaiming the DNA of amplification as mentioned above, can obtain to comprise the DNA of DNA of the present invention (A) partial nucleotide sequence.Next, the nucleotide sequence that has based on " DNA that comprises DNA of the present invention (A) partial nucleotide sequence " that obtain, design and preparation comprise the oligonucleotide of described nucleotide sequence at least about the partial nucleotide sequence of 20 Nucleotide, or comprise and the oligonucleotide of described nucleotide sequence at least about the partial nucleotide sequence complementary nucleotide sequence of 20 Nucleotide.Can obtain to comprise the DNA of partial nucleotide sequence of the DNA of the present invention (A) of 3 ' the terminal downstream of " DNA that comprises DNA of the present invention (A) partial nucleotide sequence " that extension obtains as mentioned above or 5 ' terminal upstream by carrying out PCR.PCR can use as mentioned above based on the oligonucleotide of the nucleotide sequence preparation of " DNA that comprises the partial nucleotide sequence of DNA of the present invention (A) " and comprise DNA with the carrier that is used to make up above-mentioned library insert the site adjacent area nucleotide sequence at least about the oligonucleotide of 20 Nucleotide or comprise with its nucleotide sequence complementary nucleotide sequence at least about the pairing of the oligonucleotide of 20bp as primer.PCR can for example use the chromosomal dna library for preparing from microorganism as template, and this microorganism has the ability that compound (II) is converted into compound (III) as mentioned above.By connecting this DNA that comprises DNA of the present invention (A) partial nucleotide sequence, can obtain DNA of the present invention (A).In this production method, can use commercially available test kit, as Universal Genome Walker (Clontech Company).Alternatively, full length nucleotide sequence by the DNA of the present invention (A) that obtains based on the partial nucleotide sequence that connects DNA of the present invention (A) as mentioned above prepares primer, by using this primer and by using as above-mentioned chromosomal dna library carries out PCR as template and can obtain DNA of the present invention (A).

For example, carry out PCR as template and the oligonucleotide that comprises the oligonucleotide of the former times acid sequence of nuclear shown in the SEQ ID NO:124 and comprise nucleotide sequence shown in the SEQ ID NO:129 by use as primer by using, can prepare the DNA of nucleotide sequence shown in the Nucleotide 316-1048 (partial nucleotide sequence of the nucleotide sequence of aminoacid sequence shown in the coding SEQ IDNO:159) that comprises SEQ ID NO:139 by the chromosomal DNA of black walnut streptomycete IFO 13445 preparations or chromosomal dna library.Based on the nucleotide sequence of the DNA that obtains, comprise the DNA of the nucleotide sequence that extends its 3 ' terminal downstream or 5 ' terminal upstream according to above-mentioned acquisition.By connecting the DNA that the DNA that produces can obtain to comprise nucleotide sequence shown in the SEQ ID NO:144 (nucleotide sequence that comprises the nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO:159 and the aminoacid sequence shown in the SEQ ID NO:149 of encoding).

For example, carry out PCR as template and the oligonucleotide that comprises the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:124 and comprise nucleotide sequence shown in the SEQ ID NO:129 by use as primer by using, can prepare the DNA of nucleotide sequence shown in the Nucleotide 364-1096 (partial nucleotide sequence of the nucleotide sequence of aminoacid sequence shown in the coding SEO IDNO:160) that comprises SEQ ID NO:140 by the chromosomal DNA of Tianjin island chain mould IFO 13782 preparations or chromosomal dna library.Based on the nucleotide sequence of the DNA that obtains, as described above obtains to comprise the DNA of the nucleotide sequence that extends its 3 ' terminal downstream or 5 ' terminal upstream.By connecting the DNA that the DNA that produces can obtain to comprise nucleotide sequence shown in the SEQ ID NO:145 (nucleotide sequence that comprises the nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO:150 and the aminoacid sequence shown in the SEQ ID NO:160 of encoding).

For example, carry out PCR as template and the oligonucleotide that comprises the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:124 and comprise nucleotide sequence shown in the SEQ I DNO:129 by use as primer by using, can prepare the DNA of nucleotide sequence shown in the Nucleotide 295-1027 (partial nucleotide sequence of the nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO:136) that comprises SEQ ID NO:141 by the chromosomal DNA of hot lightskyblue streptomycete IFO 14273t preparation or chromosomal dna library.Based on the nucleotide sequence of the DNA that obtains, as described above obtains to comprise the DNA of the nucleotide sequence that extends its 3 ' terminal downstream or 5 ' terminal upstream.By connecting the DNA that the DNA that produces can obtain to comprise nucleotide sequence shown in the SEQ ID NO:146 (nucleotide sequence that comprises the nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO:136 and the aminoacid sequence shown in the SEQ ID NO:151 of encoding).

For example, produce the chromosomal DNA of look streptomycete IFO 13673t preparation or chromosomal dna library by pelletizing and carry out PCR as primer by using, can prepare the DNA of nucleotide sequence shown in the Nucleotide 316-1048 (partial nucleotide sequence of the nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO:137) that comprises SEQ ID NO:142 as template and the oligonucleotide that comprises the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:124 and comprise nucleotide sequence shown in the SEQ ID NO:129 by use.Based on the nucleotide sequence of the DNA that obtains, as described above obtains to comprise the DNA of the nucleotide sequence that extends its 3 ' terminal downstream or 5 ' terminal upstream.By connecting the DNA that the DNA that produces can obtain to comprise nucleotide sequence shown in the SEQ ID NO:147 (nucleotide sequence that comprises the nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO:137 and the aminoacid sequence shown in the SEQ ID NO:152 of encoding).

For example, produce the chromosomal DNA of look streptomycete IFO 12444 preparations or chromosomal dna library by olive and carry out PCR as primer by using, can prepare the DNA of nucleotide sequence shown in the Nucleotide 316-1048 (partial nucleotide sequence of the nucleotide sequence of aminoacid sequence shown in the coding SEQID NO:138) that comprises SEQ ID NO:143 as template and the oligonucleotide that comprises the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:124 and comprise nucleotide sequence shown in the SEQ ID NO:129 by use.Based on the nucleotide sequence of the DNA that obtains, as described above obtains to comprise the DNA of the nucleotide sequence that extends its 3 ' terminal downstream or 5 ' terminal upstream.By connecting the DNA that the DNA that produces can obtain to comprise nucleotide sequence shown in the SEQ ID NO:148 (nucleotide sequence that comprises the nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO:138 and the aminoacid sequence shown in the SEQ ID NO:153 of encoding).

For example, carry out PCR as template and the oligonucleotide that comprises the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:124 and comprise nucleotide sequence shown in the SEQ ID NO:129 by use as primer by using, can prepare the DNA of nucleotide sequence shown in the Nucleotide 289-1015 (partial nucleotide sequence of the nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO:222) that comprises SEQ ID NO:232 by the redden chromosomal DNA of streptomycete IFO 13682T preparation or chromosomal dna library of rose.Based on the nucleotide sequence of the DNA that obtains, as described above obtains to comprise the DNA of the nucleotide sequence that extends its 3 ' terminal downstream or 5 ' terminal upstream.By connecting the DNA that the DNA that produces can obtain to comprise nucleotide sequence shown in the SEQ ID NO:242 (the nuclear former times acid sequence that comprises the nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO:232 and the aminoacid sequence shown in the SEQ ID NO:252 of encoding).

For example, carry out PCR as template and the oligonucleotide that comprises the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:124 and comprise nucleotide sequence shown in the SEQ ID NO:129 by use as primer by using, can prepare the DNA of nucleotide sequence shown in the Nucleotide 289-1015 (partial nucleotide sequence of the nucleotide sequence of aminoacid sequence shown in the coding SEQ IDNO:224) that comprises SEQ ID NO:234 by the chromosomal DNA of Regensburg streptomycete IFO 13446T preparation or chromosomal dna library.Based on the nucleotide sequence of the DNA that obtains, as described above obtains to comprise the DNA of the nucleotide sequence that extends its 3 ' terminal downstream or 5 ' terminal upstream.By connecting the DNA that the DNA that produces can obtain to comprise nucleotide sequence shown in the SEQ ID NO:244 (nucleotide sequence that comprises the nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO:234 and the aminoacid sequence shown in the SEQ ID NO:254 of encoding).

By according to " Molecular Cloning:A Laboratory Manual second edition " (1989), Cold Spring Harbor Laboratory Press, " Current Protocols in MolecularBiology " (1987), John Wiley ﹠amp; Described conventional genetic engineering methods such as Sons company can will be cloned in carrier by the DNA of the present invention (A) that uses above-mentioned PCR to obtain.Particularly for example, the plasmid vector in pBluescript II that can be by using plasmid vector such as Strategene company or the TA clone test kit that is contained in Invitrogen company is cloned.

In addition, for example as described belowly can prepare DNA of the present invention (A).At first, design nucleotide sequence.This nucleotide sequence coded aminoacid sequence by DNA of the present invention (A) encoded protein matter.This nucleotide sequence has at the most 60% and at least 40%, preferred 55% and at least 45% GC content at the most.The codon in the nucleotide sequence of above-mentioned protein amino acid sequence of encoding uses in from the gene of the host cell species of introducing DNA of the present invention (A) codon to use in ± 4% the scope.By have the DNA of the nucleotide sequence of design according to conventional genetic engineering method preparation, can obtain DNA of the present invention (A).

For example, can design the aminoacid sequence (SEQ ID NO:1) of code book invention protein (A1) and have at the most 55% and the nucleotide sequence of 45%GC content at least with following described method, the codon in the nucleotide sequence of the above-mentioned proteinic aminoacid sequence of wherein encoding is that codon uses in ± 4% the scope in soybean gene.At first, for example, relatively coding can use (table 22 and table 23) and soybean codon use (table 24 and table 25) available from the codon in the nucleotide sequence (SEQ IDNO:6) of protein of the present invention (A1) aminoacid sequence of abortion within the first month of pregnancy look streptomycete IFO 12898.Result based on the comparison adds nucleotide sequence shown in the SEQ ID NO:6 with nucleotide substitution so that GC content at the most 55% and at least 45% and codon to use be in soybean codon uses ± 4% scope.As this nucleotide substitution, select not cause the nucleotide substitution of amino acid replacement.For example, the encode use of leucic CTG codon is 1.22% in soybean gene and is 7.09% in nucleotide sequence shown in the SEQ IDNO:6.Equally, each that for example will be from the initial CTG codon of the Nucleotide 106,163,181,226,289,292,544,1111 and 1210 of nucleotide sequence shown in the SEQ ID NO:6 is replaced into the CTT codon; Each that will be from Nucleotide 211,547 and 1084 initial CTG codons is replaced into the CTA codon; To be replaced into the TTA codon from Nucleotide 334 initial CTG codons; Will be from Nucleotide 664,718, each in 733,772,835, the 1120 and 1141 initial CTG codons is replaced into the TTG codon; With will be replaced into the TTA codon from Nucleotide 787 initial CTG codons.So a sequence of the nucleotide sequence of design represents that in SEQ ID NO:214 wherein the use of codon shows in table 26 and table 27.In the nucleotide sequence shown in the SEQ ID NO:214, for example, the use of the leucic CTG codon of encoding is 1.71% and in soybean codon uses the scope of (1.22%) ± 4%.According to as Sambrook, J., Frisch, E.F., and Maniatis, T.; Molecular Cloning second edition, the described site-directed mutagenesis method of Cold Spring Harbor Press can be by introducing nucleotide substitution the DNA that the DNA preparation with nucleotide sequence shown in the SEQ ID NO:6 comprises nucleotide sequence shown in the SEQ ID NO:214.Alternatively, can prepare DNA by the DNA synthetic method of using PCR described in following examples 46 with nucleotide sequence shown in the SEQ ID NO:214.

Similarly, nucleotide sequence shown in the SEQ ID NO:368 is the aminoacid sequence (SEQ ID NO:222) of design code book invention protein (A23) and has at the most 55% and the example of the nucleotide sequence of 45%GC content at least that the codon use in the nucleotide sequence of the above-mentioned protein amino acid sequence of wherein encoding is in the scope of soybean gene codon use ± 4%.In addition, nucleotide sequence shown in the SEQ ID NO:393 is the aminoacid sequence (SEQID NO:224) of design code book invention protein (A25) and has at the most 55% and the example of the nucleotide sequence of 45%GC content at least that the codon use in the nucleotide sequence of the above-mentioned protein amino acid sequence of wherein encoding is in the scope of soybean gene codon use ± 4%.

According to as Sambrook, J., Frisch, E.F., and Maniatis, T.; " Molecular Cloning second edition " (1989), Cold Spring Harbor Press; " Current Protocols inMolecular Biology " (1987), John Wiley ﹠amp; The DNA of the present invention (A) that described conventional genetic engineering methods such as Sons company will so obtain is cloned in the carrier.As carrier, particularly for example, can utilize pUC 119 (TaKaRa Shuzo Company), pTVA 118N (Takara ShuzoCompany), pBluescript II (Toyobo Company), pCR2.1-TOPO (Invitrogen), pTrc99A (Pharmacia Company), pKK331-1A (Pharmacia Company), etc.

In addition, so the nucleotide sequence of the DNA of the present invention (A) that obtains can pass through F.Sanger, S.Nicklen, A.R.Coulson, described pair of deoxidation cessation method of Proceeding of National Academy of Science U.S.A. (1977) 74:5463-5467 analyzed.

The ability that compound (II) is converted into compound (III) that is used as mark with following method can confirm the ability of metabolism formula (I) PPO inhibition type herbicidal compound of the protein of the present invention (A) of DNA of the present invention (A) coding that obtained by aforesaid method.At first, as described below, described DNA is inserted carrier so that it is connected the downstream of the promotor that can work in host cell, and import host cell acquisition transformant.Next, in the presence of the electron transport system that comprises electron donor such as coenzyme NDAPH, with the culture of transformant or available from extract that destroys culture and compound (II) reaction.The reaction product of analyzing therefrom generation is with detection compound (III).So, can detect the transformant that has metabolic compounds (II) and produce the ability of compound (III), determine that this transformant has the DNA proteinic of the present invention (A) that coding has this ability.More specifically for example, the 30 μ l reaction solns that preparation is made up of 0.1M potassium phosphate buffer (pH7.0), it comprises the culture or the extract of above-mentioned transformant, β-NADPH of electron donor such as the about 2mM of final concentration, the ferredoxin that derives from spinach of the about 2mg/ml of final concentration, the ferredoxin reductase of the about 0.1U/ml of final concentration and the 3ppm compound (II) of labelled with radioisotope.With reaction solution about 30 ℃ to 40 ℃ following incubations 10 minutes to 1 hour.Behind this incubation, add 3 μ l2N HCl and 90 μ l ethyl acetate, stir and 8, centrifugal under the 000g to reclaim supernatant liquor.After the dry in a vacuum supernatant liquor, the solution that is dissolved in resistates in the ethyl acetate and on the TLC silica-gel plate, launches to obtain.By radioautographic analysis TLC plate.Be tested and appraised the ability that can confirm compound (II) is converted into compound (III) corresponding to spot with the compound (III) of labelled with radioisotope.

Use DNA of the present invention (A) or comprise described DNA partial nucleotide sequence or with the polynucleotide of partial nucleotide sequence complementary nucleotide sequence, by carry out aforesaid hybridization or PCR can further search for coding have with compound (II) be converted into compound (III) ability protein DNA or have the microorganism of this DNA.

Particularly for example, carry out the DNA of aforesaid hybridization and evaluation and probe hybridization.Use DNA of the present invention (A) or comprise DNA of the present invention (A) partial nucleotide sequence or with the polynucleotide of partial nucleotide sequence complementary nucleotide sequence as probe, hybridize with the genomic dna that derives from natural microbial, for example, described microorganism belongs to streptomyces, as abortion within the first month of pregnancy look streptomycete, brick-red streptomycete, do not produce the look streptomycete, the light gray streptomycete, Streptomyces carbophilus, grey brown streptomycete, hot lightskyblue streptomycete, the black walnut streptomycete, Tianjin island chain mould, pelletizing produces the look streptomycete, olive produces the look streptomycete, the decorative chains mould, streptomyces griseus, wool streptomycete, three damp streptomycetes, pale asphyxia streptomycete, the rose streptomycete that reddens, Shandong ground streptomycete and Regensburg streptomycete; Described microorganism belongs to saccharopolyspora strain and belongs to, as the Ta Shi saccharopolyspora strain; Deng.As the specific examples that can be used as the DNA of probe, can mention comprising SEQ ID NO:6,7,8,109,139,140,141,142,143,225,226,227,228,229,230,231, the DNA of 232, the 233 or 234 full length nucleotide sequences shown in any comprises the DNA of nucleotide sequence shown in the Nucleotide 57-730 of nucleotide sequence shown in the SEQ ID NO:6; The DNA that comprises nucleotide sequence shown in the Nucleotide 21-691 of nucleotide sequence shown in the SEQ ID NO:8; Deng.

Alternatively, can carry out PCR and the DNA that can detect amplification as mentioned above.PCR use the partial nucleotide sequence comprise DNA of the present invention (A) or with the polynucleotide of partial nucleotide sequence complementary nucleotide sequence.PCR uses the genomic dna that derives from natural microbial as template, and for example, described microorganism belongs to streptomyces, as abortion within the first month of pregnancy look streptomycete, brick-red streptomycete does not produce the look streptomycete, the light gray streptomycete, Streptomyces carbophilus, the brown streptomycete of ash, hot lightskyblue streptomycete, black walnut streptomycete, Tianjin island chain mould, pelletizing produces the look streptomycete, and olive produces look streptomycete, decorative chains mould, streptomyces griseus, the wool streptomycete, three damp streptomycetes, pale asphyxia streptomycete, the rose streptomycete that reddens, Shandong ground streptomycete and Regensburg streptomycete; Described microorganism belongs to saccharopolyspora strain and belongs to, as the Ta Shi saccharopolyspora strain; Deng.As primer, can mention based on the primer of the nucleotide sequence design of " at SEQ ID NO:6, shown in 7,8 or 109 in the nucleotide sequence the extra high zone of sequence identity " as mentioned above.As primer example more specifically, mention the pairing that comprises the primer of nucleotide sequence shown in any one among the SEQ ID NO:124 to 128 and comprise the primer of nucleotide sequence shown in the SEQ ID NO:129.

Reclaim the DNA that so detects.When the DNA that reclaims does not comprise the full length nucleotide sequence of DNA of the present invention (A), in the above described manner this DNA is used and is prepared into DNA corresponding to the full length nucleotide sequence.The DNA that obtains is imported host cell to produce transformant.Culture by the transformant that use to obtain and measure the ability that compound (II) is converted into compound (III) with aforesaid method and can assess the ability that compound (II) is converted into compound (III) by the protein of the dna encoding that imports transformant.

In order in host cell, to express DNA of the present invention (A), DNA of the present invention (A) is imported the position that it is expressed in described cell.By DNA of the present invention (A) being imported " position that can make its expression ", it is meant DNA of the present invention (A) is imported host cell so that it is in and instructs the nucleotide sequence position adjacent (promptly for example, promoting the nucleotide sequence of the RNA production of protein of the present invention (A) and code book invention protein (A)) of transcribing and translating from its nucleotide sequence.

For DNA of the present invention (A) being imported host cell so that it is positioned at the position that its is expressed, for example, wherein DNA of the present invention (A) and the promotor that works in the host cell DNA that can be operatively connected imports host cell.Here term " can be operatively connected " situation that is meant that DNA wherein of the present invention (A) is connected with promotor so that expresses under its control in promotor when importing DNA in the host cell.

When host cell is microorganism cells, as the promotor that works, for example can mention the promotor of intestinal bacteria lactose operon, the promotor of intestinal bacteria tryptophan operon, T7 phage promoter or the artificial promotor that works in intestinal bacteria are as tac promotor or trc promotor etc.In addition, can utilize the promotor that in the karyomit(e) of the microorganism that belongs to streptomyces or saccharopolyspora strain genus, was positioned at DNA of the present invention (A) upstream originally.

When host cell is vegetable cell, as the promotor that works, for example mention T-DNA deutero-constitutive promoter, as the promotor and the octopine synthase gene promotor of nopaline synthase gene; Plant virus deutero-promotor such as cauliflower mosaic virus deutero-19S and 35S promoter; The promotor of inducible promoter such as phenylalanine ammoniacalyase gene, the promotor of the promotor of chalcone synthase gene and the relevant protein gene that causes a disease; The described plant promoter of the patent publication No. 2000-166577 of Japanese unexamined.In addition, the terminator that works in vegetable cell can be connected on the promotor and DNA of the present invention (A) the quilt DNA that can be operatively connected that wherein in vegetable cell, works.In this case, preferably terminator is connected to the downstream of DNA of the present invention (A) usually.As the terminator that works, for example mention the terminator of T-DNA deutero-composing type terminator such as nopaline synthase gene (NOS); The terminator of plant virus deutero-terminator such as allium virus GV1 or GV2; At the plant terminators described in the patent publication No. 2000-166577 of Japanese unexamined; Deng.

, for example can use and containing the DNA of nucleotide sequence that coding is transported to the signal of born of the same parents' inner cell organ so that DNA is in the time of making the position that its expresses when importing DNA of the present invention (A), its be connected to DNA of the present invention (A) the upstream so that open reading-frame (ORF) in frame.It is meant and will the open reading-frame (ORF) of the sequence of the encoding transport signals of born of the same parents' inner cell organ and the open reading-frame (ORF) of DNA of the present invention (A) be connected to form a successive open reading-frame (ORF) by connecting " so that open reading-frame (ORF) is in frame ".As being provided, in born of the same parents' inner cell organ of vegetable cell, transports and localized encoding transport signals sequence in protein, for example can mention as United States Patent (USP) 5,717,084 described, as to derive from the proteinic kytoplasm precursor that is arranged in plant chloroplast encoding transport signals, the chimeric sequences that forms by the described various encoding transport signals sequences of United States Patent (USP) RE36449.More specifically, mention deriving from the soybean ribulose-1,5-bisphosphate, the chloroplast transit peptides of 5-bisphosphate carboxylase small subunit, it can obtain according to following embodiment 15 described methods.

Typically, can be with DNA of the present invention (A), connect as mentioned above and contain of the present invention DNA (A) of coding the DNA of the nucleotide sequence of the encoding transport signals of born of the same parents' inner cell organ, or this DNA and the promotor that in host cell, the works DNA that can be operatively connected wherein, can be inserted in separately in the host cell in the available carrier, this is imported host cell.When using when having had the carrier of the promotor that in host cell, works, DNA of the present invention (A) can be inserted the downstream that be present in the promotor in the carrier so that described promotor and DNA of the present invention (A) can be operatively connected.

As carrier, particularly when using intestinal bacteria as host cell, for example, can mention pUC 119 (TaKaRa Shuzo Company), pTVA 118N (Takara Shuzo Company), pBluescript II (Strategene Company), pCR2.1-TOPO (Invitrogen), pTrc99A (Amersham Pharmacia Biotech Company), pKK331-1A (AmerchamPharmacia Biotech Company), pET11d (Novagen) etc.Comprise selected marker by use and (for example give gene such as kalamycin resistance gene antibiotic resistance, neomycin resistance gene etc.) carrier, it is easily, because the phenotype of selected marker can be selected to import the transformant of DNA of the present invention as indicator.

About DNA of the present invention (A) or the carrier that comprises DNA of the present invention (A) being imported the method for host cell, when host cell is a microorganism, intestinal bacteria (E.coli) for example, subtilis (Bacillus subtilis), bacillus brevis (Bacillus brevis), pseudomonas species (Pseudomonas sp.), fermentation single cell bacterium species (Zymomonas sp.), milk-acid bacteria, acetic acid bacteria, staphylococcus species (Staphylococcus sp.), streptomyces species (Streptomyces sp.), saccharopolyspora strain species (Saccharopolyspora sp.), or yeast such as yeast saccharomyces cerevisiae (Saccharomycescerevisiae), chestnut wine fission yeast (Schizosaccaromyces ponmbe), when fungi such as Aspergillus (Aspergillus) etc., can mention at Shin Seikagaku Zikken Kouza (Nippon-Seikagaku-Kai eds., Tokyo Kagaku Dozin), 17 volumes, method described in the Biseibutu-Zikken-Hou.Alternatively, for example can use at Sambrook J., Frisch, E.F., and Maniatis, T.; " Molecular Cloning second edition " Cold SpringHarborPress (Molecular Biology, John Wiley ﹠amp; Sons, N.Y. (1989) or at " Current Protocols in Molecualr Biology " (1987), John Wiley ﹠amp; Calcium Chloride Method described in the Sons company or at " Methods in Electroporation:Gene Pulser/E.coliPulser System ", the electroporation described in the Bio-Rad Laboratories (1993).

The transformant that can select wherein to have imported DNA of the present invention (A) or comprise the carrier of DNA of the present invention (A) as indicator by the phenotype of selecting to be included in the selected marker in the carrier that inserts DNA of the present invention (A) as mentioned above for example.In addition, by from transformant, preparing DNA and carrying out at for example " Molecular Cloning second edition " Cold SpringHarbor Press (Molecular Biology, John Wiley ﹠amp with the DNA for preparing then; Sons, the described genetic engineering analytical procedure of N.Y. (1989) (as confirming restriction enzyme sites, dna sequencing, DNA hybridization, PCR etc.) can confirm whether transformant comprises DNA of the present invention (A) or contain the carrier of DNA of the present invention (A).

When host cell is vegetable cell, can mention vegetation type, for example, dicotyledons such as tobacco, cotton, Semen Brassicae campestris, beet, Arabidopis thaliana, rape (canola), flax, Sunflower Receptacle, potato, clover, lettuce, banana, soybean, pea, pod, pine tree, willow, apple, grape, orange, lemon, other citrus fruit, apricot, English walnut and other nut; Monocotyledons such as corn, paddy rice, wheat, barley, rye, oat, jowar, sugarcane and grass; Deng.Can use plant tissue, plant materials, cultured cells, seed etc. about the cell that imports DNA of the present invention (A).

About DNA of the present invention (A) or the carrier that comprises DNA of the present invention (A) being imported the method for host cell, the method of mentioning is as using agroinfection (Japanese unexamined patent publication number 2-58917 and Japanese unexamined patent publication number 60-70080), and electroporation is (Japanese unexamined patent publication number 60-251887 and Japanese unexamined patent publication number 5-68575) or particle bombardment (Japanese unexamined patent publication number 5-508316 and Japanese unexamined patent publication number 63-258525) in protoplastis.

In these cases, for example, by being selected from hygromycin phosphotransferase gene simultaneously with the carrier that comprises DNA of the present invention (A), the selected marker of neomycin phosphotransferase gene and paraxin acetyltransferase gene imports vegetable cell, can select to have imported the transformant of DNA of the present invention with the phenotype of selected marker as indicator.The selected marker can be inserted identical carrier and importing with DNA of the present invention (A).Also can import carrier that comprises the selected marker and the carrier that comprises DNA of the present invention (A) simultaneously.By with the culture medium culturing that comprises formula (I) PPO inhibition type herbicidal compound with also can select to have imported the transformant of DNA of the present invention (A) by separating wherein increasable clone.By preparation DNA from transformant with for example carry out at " Molecular Cloning second edition " Cold Spring Harbor Press (MolecularBiology, John Wiley ﹠amp with the DNA for preparing then; The genetic engineering analytical procedure of describing among the Sons, N.Y. (1989) (as confirming restriction enzyme sites, dna sequencing, DNA hybridization, PCR etc.) can confirm whether transformant comprises DNA of the present invention (A).Can remain on position in the cell the DNA in being included in nuclear with importing DNA of the present invention (A) in the vegetable cell by being inserted into the DNA that is included in born of the same parents' inner cell organ such as the chloroplast(id).

Plant transformed cell from acquisition like this, by at Shokubutu-Idenshi-Sosa-Manual:Transgenic-Shokubutu-No-Iu kurikata (Uchimiya, Kodansha-Scientific, 1990), the described culture plant cell method of 27-55 page or leaf aftergrowth body can obtain to have imported the transgenic plant of DNA of the present invention (A).In addition, by with the plant of target type with the transgenic plant mating that has imported DNA of the present invention (A) so that DNA of the present invention (A) imports in the karyomit(e) of target type plant, can produce the target vegetation type that has imported DNA of the present invention (A).

Particularly, for example, pass through Model-Shokubutu-No-Jikken-Protocol:Ine, Shiroinunazuna-Hen (Supervisors:Koh SHIMAMOTO and Kiyotaka OKADA, Shujun-sha, 1996), the described method of chapter 4 can obtain wherein to have imported the paddy rice or the Arabidopis thaliana of DNA of the present invention (A) and expression protein of the present invention (A).In addition, by importing the soybean that somatic embryos of soybean can obtain wherein to have imported DNA of the present invention (A) and express protein of the present invention (A) with particle gun according to the described method of the patent publication No. 3-291501 of Japanese unexamined.Equally, by according to Fromm, M.E. waits Bio/Technology, and 8; The 838th page of (1990) described method imports the corn that the corn somatic embryo can obtain wherein to have imported DNA of the present invention (A) and express protein of the present invention (A) with particle gun.By according to TAKUMI etc., Journal ofBreeding Society (1995), the wheat prematurity scutellum that 1, the 57 page of described ordinary method of 44:Extra volume imports sterile culture with particle gun with gene can obtain wherein to have imported the wheat of DNA of the present invention (A) and expression protein of the present invention (A).Equally, by according to HAGIO, etc., Journal of Breeding Society (1995), 44; 1, the 67 page of described ordinary method of Extra volume can obtain wherein to have imported the barley of DNA of the present invention (A) and expression protein of the present invention (A) with the barley prematurity scutellum of particle gun importing sterile culture.

By described herbicidal compound being changed into the compound of low weeding activity in its cell, the transformant that has wherein imported DNA of the present invention (A) and expression protein of the present invention (A) can alleviate the plant injury that is caused by compound (I).Particularly for example, be dispersed in the cultivation of plants zone that expectation is cultivated by the microorganism that will express protein of the present invention (A) before the sowing of expectation plant, the herbicidal compound that remains in the soil can be alleviated the damage to the expectation plant by metabolism.In addition, by making the various expression of plants protein of the present invention (A) of expectation, give the ability that described plant is metabolized to the PPO inhibition type herbicidal compound of formula (I) SA compound.As a result, alleviate in the plant, give resistance described compound from the plant injury of herbicidal compound.

For example the cell that comprises DNA of the present invention (A) by cultivation can prepare protein of the present invention (A).About this cell, mention the microorganism of expressing DNA of the present invention (A) and having the ability of production protein of the present invention (A), as from the isolating microorganism strains that comprises DNA of the present invention (A) of nature, by handling by natural bacterial strain deutero-mutant strain with reagent or UV treatment etc.More specifically for example, mention the microorganism that belongs to streptomyces, abortion within the first month of pregnancy look streptomycete IFO 12898, brick-red streptomycete ATCC 21469 does not produce look streptomycete IFO 12735, light gray streptomycete ATCC11796, Streptomyces carbophilus SANK62585, the brown streptomycete IFO 12870t of ash, hot lightskyblue streptomycete IFO 14273t, black walnut streptomycete IFO13445, Tianjin island chain mould IFO13782, pelletizing produces look streptomycete IFO 13673t, and olive produces look streptomycete IFO 12444, decorative chains mould IFO 13069t, streptomyces griseus ATCC 10137, streptomyces griseus IFO 13849T, wool streptomycete IFO 12787T, three damp streptomycete IFO 13855T, pale asphyxia streptomycete IFO 13434T, the rose streptomycete IFO 13682T that reddens, Shandong ground streptomycete IFO 15875T and Regensburg streptomycete IFO13446T, etc.; Or belong to the microorganism that saccharopolyspora strain belongs to, as Ta Shi saccharopolyspora strain JCM 9383t etc.In addition, can mention the transformant that has wherein imported DNA of the present invention (A) or comprised the carrier of DNA of the present invention (A).Particularly for example, mention wherein will with the tac promotor, the trc promotor, the DNA of the present invention (A) that lac promotor or t7 phage promoter can be operatively connected imports colibacillary transformant.As more specific examples, mention the e. coli jm109/pKSN657 that describes in the following embodiments, e. coli jm109/pKSN657F, e. coli jm109/pKSN923, e. coli jm109/pKSN923F, e. coli jm109/pKSN11796, e. coli jm109/pKSN11796F, e. coli jm109/pKSN671, e. coli jm109/pKSN671F, e. coli jm109/pKSNCA, e. coli jm109/pKSN646, e. coli jm109/pKSN646F, e. coli jm109/pKSN849AF, e. coli jm109/pKSN1618F, e. coli jm109/pKSN474F, e. coli jm109/pKSN1491AF, e. coli jm109/pKSN1555AF, e. coli jm109/pKSN1584F, e. coli jm109/pKSN1609F etc.

As cultivating the above-mentioned substratum that comprises the microorganism of DNA of the present invention (A), can use any in those that are generally used for culturing micro-organisms, it comprises carbon source and nitrogenous source, if suitable organic and inorganic salt.The compound that can add the protoheme precursor is as amino-laevulic acid.

As carbon source, for example mention carbohydrate such as glucose, fructose, sucrose and dextrin; Sugar alcohol such as glycerine and Sorbitol Powder; With organic acid such as fumaric acid, citric acid and pyruvic acid; Deng.Above listed carbon source add substratum amount normally based on about 0.1% (w/v) of substratum total amount to about 10% (w/v).

As nitrogenous source, for example mention the ammonium salt such as the ammonium chloride of mineral acid, ammonium sulfate and ammonium phosphate; Organic acid ammonium salt such as fumaric acid ammonium and ammonium citrate; Organic nitrogen source such as meat extract, yeast extract, wort, soyflour, corn steep liquor, cottonseed meal, dry yeast, casein hydrolysate; And amino acid.Above listed in those, organic acid ammonium salt, organic nitrogen source and amino acid also can be used as carbon source usually.The amount that adds nitrogenous source normally based on about 0.1% (w/v) of substratum total amount to about 10% (w/v).

As inorganic salt, for example, mention phosphoric acid salt such as potassiumphosphate, dipotassium hydrogen phosphate, sodium phosphate, Sodium phosphate dibasic; Muriate such as Repone K, sodium-chlor, cobalt chloride hexahydrate; Vitriol such as sal epsom, ferric sulfate heptahydrate, zinc sulfate heptahydrate, manganous sulfate trihydrate; Deng.The amount that adds normally based on about 0.0001% (w/v) of substratum total amount to about 1% (w/v).

Be connected in the situation of transformant of DNA in lac UV5 promotor downstream at the nuclear former times acid sequence that cultivate to keep being connected in the DNA of the present invention (A) in T7 phage promoter downstream and the t7 rna polymerase of wherein encoding (λ DE3 lysogenic bacteria), typically can add on a small quantity isopropyl-(hereinafter referred to as " IPTG ") is for example induced protein of the present invention (A) as inductor production.Imported in the transformant situation of DNA in cultivation, also IPTG can have been added substratum, a DNA of the present invention (A) and a class are by lactose-induced promotor such as tac promotor among the described DNA, and trc promotor and lac promotor can be operatively connected.

Can comprise the liquid phase cultivation as the rotational oscillation cultivation according to the method that is generally used for culturing micro-organisms, reciprocating type shaking culture, small-sized fermentation (cultivation of small-sized fermentation jar) and jar cultivation; Or the solid phase cultivation, cultivate the microorganism that comprises DNA of the present invention (A).When using the small-sized fermentation jar, with per minute about 0.1 to the rate of venting of about 2 times of nutrient solution volumes sterile air is imported the small-sized fermentation jar usually.The temperature of implementing to cultivate can change in the scope that allows microorganism growth, is generally about 15 ℃ to about 40 ℃, and the pH of substratum is about 6 to about 8.Incubation time can change according to culture condition, is about 1 day to about 10 days usually.

The protein of the present invention (A) of the microorganisms producing by comprising DNA of the present invention (A) can be used for the PPO inhibition type herbicidal compound of processing formula (I) with various forms, culture as the microorganism of production protein of the present invention (A), the microorganism cells of production protein of the present invention (A), by handling the material that this cell obtains, the cell-free extract of microorganism, the complete protein of purifying not, the protein of purifying etc.The above-mentioned material that obtains by the processing cell comprises for example freeze drying cell, acetone drying cell, ground cell, the autolyzate of cell, the cell of supersound process, the cell of alkaline purification, the cell that organic solvent is handled etc.Alternatively, can be adsorbed on inorganic carrier such as silica gel or stupalith according to currently known methods such as use, polysaccharide derivates such as DEAE-Mierocrystalline cellulose, synthetic polymer such as Amberite IRA-935 (trade(brand)name, by Rohm and Haas preparation) etc. on the carrier combined techniques, be included in polymkeric substance such as polyacrylamide with use, sulfur-bearing polysaccharide gel (for example carrageenan gel), alginic acid glue, the method that comprises in the pseudostructure of agaropectin etc. fixes any protein of the present invention (A) in the above-mentioned various forms, is used to handle above-mentioned herbicidal compound then.

As the method for purifying protein of the present invention (A) from the culture of the microorganism that comprises DNA of the present invention (A), can use routine to be used for method for purifying proteins.For example can mention following method.

At first, by harvested cell from the culture of microorganism such as centrifugal grade, then by supersound process, DYNOMILL handles, and physics such as FRENCH PRESS processing break, or by using tensio-active agent or lysis enzyme such as N,O-Diacetylmuramidase chemical disruption cell.From the lysate of the generation of acquisition like this, by centrifugal, membrane filtrations etc. are removed insolubles with the preparation cell-free extract, it is then by any suitable separation and purification process, as cation-exchange chromatography, anion-exchange chromatography, hydrophobic chromatography, classifications such as gel permeation chromatography, purifying protein of the present invention (A) thus.The support material that is used for this chromatography for example comprise resin carrier as with carboxymethyl (CM), diethyllaminoethyl (DEAE), Mierocrystalline cellulose, dextran and agarose that phenyl or butyl connect.Also can use commercially available pillar of having filled any carrier, as Q-Sepharose FF, Phenyl-Sepharose HP, PD-10 and HiLoad26/10Q Sepharose HP (trade(brand)name, from Amersham Pharmacia Biotech), TSK-gel G3000SW (trade(brand)name, TOSOH CORPORATION).

An example of purifying protein of the present invention (A) is provided.

Microorganism cells by centrifugal results generation protein of the present invention (A) is suspended in damping fluid such as the 0.1M potassiumphosphate (pH7.0) then.The supersound process suspension is with ruptured cell, and about 40, about 30 minutes of the lysate of the generation of centrifugal acquisition like this is obtaining supernatant liquor under the 000g, and it is then 150, under the 000g centrifugal about 1 hour to reclaim supernatant liquor (cell-free extract).With the cell-free extract that obtains carry out the ammonium sulfate fractional separation with obtain solubilized in the presence of the 45%-saturated ammonium sulphate and under the saturated ammonium sulfate of 55%-sedimentary fraction.Behind the solvent that uses PD10 post (AmershamPharmacia Biotech Company) with damping fluid that does not comprise ammonium sulfate such as 1M potassiumphosphate exchange fraction, with sample on the fraction that produces to HiLoad 26/10 Q SepharoseHP post (Amersham Pharmacia Biotech Company) for example.Obtain a series of eluate fractions with two three propane (bistripropane) of 20mM and NaCl linear gradient elution pillar.Recovery is presented at the active fraction that under the existence of the electron transport system that comprises electron donor such as coenzyme NADP 11 compound (II) is converted into compound (III).Next, by after using the damping fluid in the exchange of PD10 post (Amersham Pharmacia Biotech Company) for example fraction, to Bio-Scale Ceramic, hydroxylapatite for example is on the I type pillar CHT10-I (BioRad Company) with sample on the fraction that reclaims.(comprising 1.5mM CaCl with buffer A ₂The 2mM potassium phosphate buffer; PH7.0) after the washing pillar, (comprise 0.03mM CaCl with the buffer B of buffer A and linear gradient ₂The 100mM potassium phosphate buffer) the wash-out pillar to be to obtain a series of eluate fractions.Recovery is presented at the active fraction that under the existence of the electron transport system that comprises electron donor such as coenzyme NADP 11 compound (II) is converted into compound (III).By after using the damping fluid in the exchange of PD10 post (Amersham Pharmacia Biotech Company) for example fraction, by for example ultrafiltration (microcon filter device microcon-30; Millipore company) concentrates the fraction that reclaims.The fraction that produces for example is expelled to HiLoad 16/60 Superdex post 75pg post (Amersham Pharmacia Biotech Company) neutralization obtains a series of eluate fractions with comprising the 0.05M potassium phosphate buffer wash-out of 0.15M NaCl (pH7.0).Recovery is presented at the active fraction that under the existence of the electron transport system that comprises electron donor such as coenzyme NADP 11 compound (II) is converted into compound (III).As required can purifying protein of the present invention (A) by separating with SDS-PAGE.

By with aforesaid method purifying protein of the present invention (A), then the protein of the present invention (A) that obtains is used as immunizing antigen, can produce the antibody (hereinafter being sometimes referred to as " antibody of the present invention (A) ") of identification protein of the present invention (A).

Particularly for example, in order to the protein of the present invention (A) of aforesaid method purifying as antigen-immunized animal.For example, for immune animal such as mouse, hamster, cavy, chicken, rat, rabbit, dogs etc. are used at for example W.H.Newsome, and administration of antigens is once at least for the routine immunization method of describing among J.Assoc.Off.Anal.Chem.70 (6) 1025-1027 (1987).As drug dosage schedule, for example mention at 7 to 30 days interval preferred 12 to 16 days interval administration 2 or 3 times.Its dosage is for example each about 0.05mg-2mg antigen of administration.Route of administration can be selected from subcutaneous administration, intradermal administration, and the intraperitoneal administration, intravenous administration, and intramuscular administration, intravenously, intraperitoneal or subcutaneous injection are typical administering modes.Typically antigen is used in the back in being dissolved in suitable damping fluid; described damping fluid is sodium phosphate buffer or physiological saline for example; it comprises at least a normally used adjuvant such as complete Freund's adjuvant (Aracel A; the mixture of Bayol F and fast knot nuclear bacillus (tubercule bacillus)), RAS[MPL (single phosphoryl lipoid A)+TDM (synthetic trehalose dicorynomycolate)+CWS (cell wall skeleton) adjuvant system] or aluminium hydroxide.Yet, depend on route of administration or illness, can not use above-mentioned adjuvant." adjuvant " is non-specific at antigenic immunoreactive material through strengthening after the antigen administration.After the animal 0.5-4 that feeds administration of antigens month, sampling small amounts of blood and measurement antibody titer from the ear vein of for example animal.When antibody titer improves, according to the situation suitable number of times of administration of antigens again.For example, can use antigen again with the dosage of about 100 μ g-1000 μ g.Blood is collected in administration 1 or after 2 months the last time in due form from immune animal.By routine techniques as by centrifugal or with ammonium sulfate or use polyethylene glycol precipitation, chromatography such as gel permeation chromatography, ion exchange chromatography and affinity chromatography wait the blood classification, can obtain the antibody of the present invention (A) as polyclonal antiserum.In addition, can be with antiserum(antisera) for example 56 ℃ of following incubations 30 minutes so that the complement system inactivation.

Alternatively, chemosynthesis comprises the polypeptide of protein of the present invention (A) partial amino-acid series and is applied to animal as immunizing antigen, produces antibody of the present invention (A) thus.About the amino acid sequence of polypeptide of using as immunizing antigen, from the aminoacid sequence of protein of the present invention (A), select to have the aminoacid sequence of low homology as far as possible with other proteinic aminoacid sequence.Polypeptide that form by selected aminoacid sequence by the ordinary method chemosynthesis, 10 amino acid to 15 amino acid lengths, and for example use MBS etc. crosslinked with carrier proteins such as Limulus plyhemus hemocyanin, be used for immune animal such as rabbit then as mentioned above.

Then the antibody of the present invention (A) that produces is contacted the complex body by protein and above-mentioned antibody in the routine immunization method test samples then, thereby the protein of the present invention (A) in the test samples or comprise the polypeptide of its partial amino-acid with sample.Particularly for example, be evaluated at the existence of checking protein of the present invention (A) in the sample or quantitative protein of the present invention (A) as western blot analysis that can the application of the invention antibody (A) as described in following examples 45 or 73.

In addition, for example, according to routine immunization method, by antibody of the present invention (A) is contacted with the test cell or by the sample of testing cell preparation, then detect above-mentioned antibody and test cell or, can detect the cell of expressing protein of the present invention (A) by the proteinic complex body in the sample of test cell preparation.By the cell of detection expression protein of the present invention like this (A), can also from various kinds of cell, select to express the cell of protein of the present invention (A).Use antibody of the present invention (A) can also clone or select to comprise the clone of protein of the present invention (A).For example, by extract genomic dna from the cell of expressing protein of the present invention (A), then genomic dna is inserted expression vector can produce genomic library.With the genomic library transfered cell.From the groups of cells that obtains, use antibody of the present invention (A) to select to express the cell of protein of the present invention (A) with aforesaid method.

The test kit that comprises antibody of the present invention (A) can be used for detect as mentioned above protein of the present invention (A) or analyze the cell of detection or searching expression protein of the present invention (A).Test kit of the present invention can comprise the required reagent of above-mentioned analytical procedure except antibody of the present invention (A), can contain the reagent that uses with antibody of the present invention (A).

By with the PPO inhibition type herbicidal compound of formula (I) in the presence of the electron transport system that comprises electron donor such as coenzyme NADP 11 with protein of the present invention (A) reaction, with the above-claimed cpd metabolism with change into the compound that hangs down weeding activity.Particularly for example, by in the presence of the electron transport system that comprises electron donor such as coenzyme NADP 11 compound (II) and protein of the present invention (A) being reacted, compound (II) is converted to compound (III), and it shows does not have weeding activity basically.The example of protein in this case (A) is protein of the present invention (A).A variant of protein of the present invention (A) can be used, a plurality of variants can be used together.

The compound of formula (I) is the compound with uridylic structure.As concrete example, can mention any one compound in compound (II) or the formula (IV) to (IX) (hereinafter being called compound (IV)) to compound (IX).According to can synthetic compound (II) and compound (IX) in method described in the patent publication No. 2000-319264 of Japanese unexamined, can synthetic compound (IV) and compound (V) according to United States Patent (USP) 5183492 described methods, can synthetic compound (VI) according to United States Patent (USP) 5674810 described methods, can synthetic compound (VII) according to the described method of Japanese unexamined patent publication number 3-204865, can synthetic compound (VIII) according to the described method of Japanese unexamined patent publication number 6-321941.

In addition, as the specific examples of formula (I) compound, can mention in the formula (X) to (XVII) compound (below be called compound (X)) of any one to compound (XVII).

By making compound be present in reaction, radioisotopic compound of mark (II) and protein of the present invention (A) reaction in the presence of the electron transport system that comprises electron donor such as coenzyme NADP 11 in the described reaction, with the compound (II) that detects mark as a token of to the conversion reaction of the compound (III) of mark by the competitive inhibition of protein of the present invention (A), can select can be the compound of metabolic reaction substrate of protein of the present invention (A).When measuring, typically test compound is added to the amount of the volumetric molar concentration of 1-100 times of tagged compound (II) from the existing of the competitive inhibition of test compound.

Can be for example at the salt that comprises mineral acid such as alkali metal phosphate such as sodium phosphate and potassiumphosphate; Or carry out the reaction of compound (I) and protein of the present invention (A) in the water-containing buffering liquid of organic acid salt such as alkali-metal acetate such as sodium acetate and potassium acetate etc.Typically be about at the most 30% (w/v) and preferred about 0.001% (w/v) to 20% (w/v) in metabolic reaction liquid Chinese style (I) compound concentrations.Comprising the electron transport system of electron donor such as NADPH or the amount of protein of the present invention (A) can for example change according to the reaction times.Temperature of reaction is selected from typically about 10 ℃-70 ℃ scope, preferred about 20 ℃-50 ℃.The pH of reaction solution is selected from the typically scope of about 4-12, preferably about 5-10.Reaction times can change as required, typically about 1 hour to 10 days.

In addition, can in the cell that comprises DNA of the present invention (A), carry out the reaction of compound (I) and protein of the present invention (A).As the cell that comprises DNA of the present invention (A), for example mention and have the microorganism of expressing DNA of the present invention (A) and production protein of the present invention (A), as from the isolating bacterial strain that comprises those microorganisms of DNA of the present invention (A) of occurring in nature, by be derived from the mutant strain of this microorganism strains with chemical or UV treatment, wherein DNA of the present invention (A) or the carrier that comprises DNA of the present invention (A) are imported the microorganism transformed cell of host cell.In addition, mention plant transformed cell that imports DNA of the present invention (A) or the cell that imports the conversion plant of DNA of the present invention (A).In this case, formula (I) compound directly can be added to the cell that comprises DNA of the present invention (A) maybe can add to cell culture medium or with the soil of cells contacting in so that enter cell.The electron transport system that comprises electron donor such as NADPH can be present in originally in the cell system and can add from outside.

For example by detecting the metabolism that the compound that is produced by compound (I) metabolism can confirm the compound (I) that caused by protein of the present invention (A).Particularly for example, as mentioned above can be with the compound (III) that HPLC analyzes or the TLC analyzing and testing is produced by metabolic compounds (II).

In addition, based on the weeding activity in compound (I) and protein of the present invention (A) reaction afterreaction solution compare be lower than compound (I) not with the situation of protein of the present invention (A) reaction, can confirm the metabolism of the compound (I) that causes by protein of the present invention (A).Method as the check weeding activity, for example mention and wherein above-mentioned reaction soln is applied to weeds such as barnyard grass (Echinochloacrus-galli), big fringe amur foxtail (Alopercurus myosuroides), the method for Ivyleaf morningglory (Ipomoea hederacea) and piemarker (Abutilon theophrasti) and inspection herbicidal effect; Or wherein weeds are being used the method for cultivating and check herbicidal effect on the pedotheque of above-mentioned reaction soln; Deng.In addition, mentioning wherein can be with above-mentioned reaction soln point on the leaf dish of taking from plant and check the method for the existence of the plant injury (bleaching) that is caused by reaction soln.

In addition, serve as a mark by detection, the PPO in compound (I) and the reacted reaction soln of protein of the present invention (A) suppress activity compare be lower than compound (I) wherein not with the reaction soln of protein of the present invention (A) reaction in activity, can confirm the metabolism of the compound (I) that protein of the present invention (A) causes.PPO is the enzyme that catalysis protoporphyrinogen IX transforms to protoporphyrin IX (hereinafter referred to as " PPIX ").For example, after adding above-mentioned reaction soln in the PPO reactive system, the substrate-protoporphyrinogen IX that adds PPO is also in the dark at 30 ℃ of about 1-2 of following incubation hours.Subsequently, use the amount of measurement PPIX in the solution of each incubation such as HPLC.When the amount of PPIX in the system that is adding compound (I) and the reacted reaction soln of protein of the present invention (A) surpass adding compound (I) not with the system of the reacted reaction soln of protein of the present invention (A) in the PPIX amount time, determine that compound (I) is by protein of the present invention (A) metabolism.As PPO, can use the protein of purifying from plant etc. or from the chloroplast(id) fraction of plant.When using the timesharing of chloroplast(id) level, amino-laevulic acid rather than protoporphyrinogen IX can be used for the PPO reactive system.Amino-laevulic acid is the precursor of protoporphyrinogen IX in chlorophyll-protoheme biosynthetic pathway.Provide an example more specifically below among the embodiment 42.

By like this and protein of the present invention (A) reaction, can carry out the processing of formula (I) PPO inhibition type herbicidal compound, it causes the compound metabolism and changes into the compound of low weeding activity.Can alleviate plant injury by the processing of on the plant growing zone, spraying described compound from described compound, particularly for example, be sprayed on the plant growing zone and be retained in compound and protein of the present invention (A) reaction among plant residue or the soil etc.

Make compound (I) and " electron transport system that comprises electron donor " that protein of the present invention (A) reacts as being used for, for example can mention comprising NADPH, ferredoxin and ferredoxin-NADP ⁺The system of reductase enzyme.

As the method that in system, provides " electron transport system that comprises electron donor " that compound (I) and protein of the present invention (A) are reacted, for example mention and to derive from the NADPH of plant such as spinach, ferredoxin and the ferredoxin-NADP that derives from plant such as spinach ⁺Reductase enzyme adds the method for above-mentioned reactive system.In addition, can with can from microorganism as preparation the intestinal bacteria, comprise fraction for the effective composition of electron transport system in protein of the present invention (A) reaction system be added to as described in the reaction system.In order to prepare this fraction, for example by centrifugal grade from the culture of microorganism after the harvested cell, by supersound process, DYNOMILL handles, and physics such as FRENCH PRESS processing break or pass through to use tensio-active agent or lysis enzyme such as N,O-Diacetylmuramidase chemical disruption cell.From the lysate of the generation of acquisition like this, by centrifugal, membrane filtration etc. are removed insolubles with the preparation cell-free extract.Cell-free extract can be used to exchange above-mentioned ferredoxin as the fraction that comprises for the effective composition of electron transport system in the reaction system of protein of the present invention (A).In addition, when the system that electronics can be delivered to protein of the present invention (A) from electron donor is present in this cell, reaction as protein of the present invention (A) wherein and compound (I) is the situation of carrying out in cell such as microorganism or vegetable cell, can not add electron transport system again.

As ferredoxin, for example can use the ferredoxin (hereinafter being called " protein of the present invention (B) " sometimes jointly) that derives from microorganism, described microorganism belongs to streptomyces, as abortion within the first month of pregnancy look streptomycete, brick-red streptomycete, do not produce the look streptomycete, the light gray streptomycete, hot lightskyblue streptomycete, the black walnut streptomycete, Tianjin island chain mould, pelletizing produces the look streptomycete, olive produces look streptomycete, decorative chains mould, streptomyces griseus, the wool streptomycete, three damp streptomycetes, pale asphyxia streptomycete, the rose streptomycete that reddens, Shandong ground streptomycete and Regensburg streptomycete, more specifically, abortion within the first month of pregnancy look streptomycete IFO 12898, brick-red streptomycete ATCC 21469, do not produce look streptomycete IFO 12735, light gray streptomycete ATCC11796, hot lightskyblue streptomycete IFO 14273t, black walnut streptomycete IFO 13445, Tianjin island chain mould IFO 13782, pelletizing produces look streptomycete IFO 13673t, and olive produces look streptomycete IFO 12444, decorative chains mould IFO 13069t, streptomyces griseus ATCC 10137, streptomyces griseus IFO 13849T, wool streptomycete IFO 12787T, three damp streptomycete IFO 13855T, pale asphyxia streptomycete IFO13434T, the rose streptomycete IFO 13682T that reddens, Shandong ground streptomycete IFO 15875T and Regensburg streptomycete IFO 13446T, etc.; Or belong to the microorganism that saccharopolyspora strain belongs to, as the Ta Shi saccharopolyspora strain, more specifically, Ta Shi saccharopolyspora strain JCM 9383t etc.Particularly for example, can mention the ferredoxin (hereinafter being sometimes referred to as " protein of the present invention (B) ") that is selected from following combination.

＜protein combination 〉

(B1) comprise the protein (hereinafter being sometimes referred to as " protein of the present invention (B1) ") of aminoacid sequence shown in the SEQ ID NO:12;

(B2) comprise the protein (hereinafter being sometimes referred to as " protein of the present invention (B2) ") of aminoacid sequence shown in the SEQ ID NO:13;

(B3) comprise the protein (hereinafter being sometimes referred to as " protein of the present invention (B3) ") of aminoacid sequence shown in the SEQ ID NO:14;

(B4) comprise the protein (hereinafter being sometimes referred to as " protein of the present invention (B4) ") of aminoacid sequence shown in the SEQ ID NO:111;

(B5) comprise NO:12 with SEQ ID, SEQ ID NO:13, aminoacid sequence shown in any one has the ferredoxin of the aminoacid sequence of at least 80% sequence identity among SEQ ID NO:14 or the SEQID NO:111;

(B6) comprise NO:12 with coding SEQ ID, SEQ ID NO:13, the nucleotide sequence of aminoacid sequence shown in any one has the ferredoxin of the coded aminoacid sequence of the nucleotide sequence of at least 90% sequence identity among SEQ ID NO:14 or the SEO ID NO:111;

(B7) comprise the protein (hereinafter being sometimes referred to as " protein of the present invention (B7) ") of aminoacid sequence shown in the SEQ ID NO:149;

(B8) comprise the protein (hereinafter being sometimes referred to as " protein of the present invention (B8) ") of aminoacid sequence shown in the SEQ ID NO:150;

(B9) comprise the protein (hereinafter being sometimes referred to as " protein of the present invention (B9) ") of aminoacid sequence shown in the SEQ ID NO:151;

(B10) comprise the protein (hereinafter being sometimes referred to as " protein of the present invention (B10) ") of aminoacid sequence shown in the SEQ ID NO:152;

(B11) comprise the protein (hereinafter being sometimes referred to as " protein of the present invention (B11) ") of aminoacid sequence shown in the SEQ ID NO:153;

(B12) a kind of ferredoxin, its comprise with at SEQ ID NO:149, SEQ IDNO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:245, SEQ IDNO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ IDNO:251, the aminoacid sequence of any one expression among the SEQ ID NO:253 have at least 80% sequence identity aminoacid sequence or with at SEQ ID NO:150, any of the aminoacid sequence of representing among SEQ ID NO:252 or the SEQID NO:254 has the aminoacid sequence of at least 90% sequence identity;

(B13) a kind of ferredoxin, it comprises the NO:149 with coding SEQ ID, SEQ IDNO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ IDNO:245, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ IDNO:250, SEQ ID NO:251, SEQ ID NO:252, any one nucleotide sequence of aminoacid sequence has the nucleotide sequence coded aminoacid sequence of at least 90% sequence identity shown in SEQ ID NO:253 or the SEQ IDNO:254;

(B14) comprise the protein of aminoacid sequence shown in the SEQ ID NO:245;

(B15) comprise the protein of aminoacid sequence shown in the SEQ ID NO:247;

(B16) comprise the protein of aminoacid sequence shown in the SEQ ID NO:248;

(B17) comprise the protein of aminoacid sequence shown in the SEQ ID NO:249;

(B18) comprise the protein of aminoacid sequence shown in the SEQ ID NO:250;

(B19) comprise the protein of aminoacid sequence shown in the SEQ ID NO:251;

(B20) comprise the protein of aminoacid sequence shown in the SEQ ID NO:252;

(B21) comprise the protein of aminoacid sequence shown in the SEQ ID NO:253; With

(B24) comprise the protein of aminoacid sequence shown in the SEQ ID NO:254.

According at Molecular Cloning:A Laboratory Manual second edition (1989), ColdSpring Harbor Laboratory Press; Current Protocols in Molecular Biology (1987), John Wiley ﹠amp; The conventional genetic engineering method of describing in the Sons company etc. is based on being coded in SEQ ID NO:12,13,14,111,149,150,151,152,153,245,247,248,249,250, the nucleotide sequence of the aminoacid sequence of the protein of the present invention (B) of expression can obtain the DNA (hereinafter being sometimes referred to as " DNA of the present invention (B) ") of code book invention protein (B) in 251,252,253 or 254.

DNA (hereinafter being called " DNA of the present invention (B) " sometimes jointly) as code book invention protein (B) mentions

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (B1) ") of aminoacid sequence shown in the SEQ ID NO:12;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (B2) ") of aminoacid sequence shown in the SEQ ID NO:13;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (B3) ") of aminoacid sequence shown in the SEQ ID NO:14;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (B4) ") of aminoacid sequence shown in the SEQ ID NO:111;

Coding comprises the NO:12 with SEQ ID, SEQ ID NO:13, and aminoacid sequence shown in any one has the DNA of ferredoxin of the aminoacid sequence of at least 80% sequence identity among SEQ ID NO:14 or the SEQ IDNO:111;

The DNA of coding ferredoxin, described ferredoxin comprises the NO:12 with coding SEQ ID, SEQ ID NO:13, the coded aminoacid sequence of nucleotide sequence that the nucleotide sequence of aminoacid sequence has at least 90% sequence identity shown in any one among SEQIDNO:14 or the SEQ ID NO:111;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (B7) ") of aminoacid sequence shown in the SEQ ID NO:149;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (B8) ") of aminoacid sequence shown in the SEQ ID NO:150;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (B9) ") of aminoacid sequence shown in the SEQ ID NO:151;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (B10) ") of aminoacid sequence shown in the SEQ ID NO:152;

Coding comprises the protein DNA (hereinafter being sometimes referred to as " DNA of the present invention (B11) ") of aminoacid sequence shown in the SEQ ID NO:153;

The DNA of coding ferredoxin, described ferredoxin comprise with at SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:245, SEQID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ IDNO:251, the aminoacid sequence of any one expression among the SEQ ID NO:253 have at least 80% sequence identity aminoacid sequence or with at SEQ ID NO:150, the aminoacid sequence that the aminoacid sequence of any one expression has at least 90% sequence identity among SEQ ID NO:252 or the SEQID NO:254;

The DNA of coding ferredoxin, described ferredoxin comprises the IDNO:149 with coding SEQ, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ IDNO:153, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:248, SEQ IDNO:249, SEQ ID NO:250, SEQ ID NO:251, the nucleotide sequence coded aminoacid sequence that the nucleotide sequence of SEQ ID NO:252, SEQ ID NO:253 or SEQ ID NO:254 aminoacid sequence shown in any has at least 90% sequence identity;

Coding comprises the protein DNA of aminoacid sequence shown in the SEQ ID NO:245;

Coding comprises the protein DNA of aminoacid sequence shown in the SEQ ID NO:247;

Coding comprises the protein DNA of aminoacid sequence shown in the SEQ ID NO:248;

Coding comprises the protein DNA of aminoacid sequence shown in the SEQ ID NO:249;

Coding comprises the protein DNA of aminoacid sequence shown in the SEQ ID NO:250;

Coding comprises the protein DNA of aminoacid sequence shown in the SEQ ID NO:251;

Coding comprises the protein DNA of aminoacid sequence shown in the SEQ ID NO:252;

Coding comprises the protein DNA of aminoacid sequence shown in the SEQ ID NO:253; With

Coding comprises the protein DNA of aminoacid sequence shown in the SEQ ID NO:254.

As the example more specifically of DNA of the present invention (B), can mention comprising SEQ ID NO:15,16,17,112,154,155,156,157,158,255,257,258,259,260,261, the DNA of nucleotide sequence shown in any one or comprise the IDNO:15 with SEQ, 16 in 262,263 or 264,17,112,154,155,156,157,158,255,257,258,259,260, nucleotide sequence shown in any one has the DNA of the nucleotide sequence of at least 90% sequence identity in 261,262,263 or 264.

According in condition described in the method for preparing DNA of the present invention (A), carry out the method for PCR or wherein this DNA can be prepared this DNA as the hybridizing method of probe as primer with the DNA of the partial nucleotide sequence that comprises its nucleotide sequence by implementing.

Particularly for example, by using from the chromosomal DNA of abortion within the first month of pregnancy look streptomycete IFO 12898 preparations or chromosomal dna library as template, carry out PCR with the oligonucleotide that comprises nucleotide sequence shown in the SEQ ID NO:53 as primer with the oligonucleotide that comprises nucleotide sequence shown in the SEQ ID NO:105 by use and can prepare the DNA that comprises the nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO:12, or comprise the DNA of nucleotide sequence shown in the SEQ ID NO:15.

In addition, by using from the chromosomal DNA of Ta Shi saccharopolyspora strain JCM 9383t preparation or chromosomal dna library as template, carry out PCR with the oligonucleotide that comprises nucleotide sequence shown in the SEQ ID NO:63 as primer with the oligonucleotide that comprises nucleotide sequence shown in the SEQ ID NO:106 by use and can prepare the DNA that comprises the nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO:13, or comprise the DNA of nucleotide sequence shown in the SEQ ID NO:16.

In addition, by using from the chromosomal DNA of brick-red streptomycete ATCC 21469 preparations or chromosomal dna library as template, carry out PCR with the oligonucleotide that comprises nucleotide sequence shown in the SEQ ID NO:72 as primer with the oligonucleotide that comprises nucleotide sequence shown in the SEQ ID NO:107 by use and can prepare the DNA that comprises the nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO:14, or comprise the DNA of nucleotide sequence shown in the SEQ ID NO:17.

In addition, by with by comprising coding SEQ ID NO:12,13,14,111,149, the DNA that at least 20 Nucleotide of pact of the nucleotide sequence of aminoacid sequence shown in any one are formed in 150,151,152 or 153 is as probe, with chromosomal dna library hybridization, then detection and recovery and described probe specificity bonded DNA can obtain DNA of the present invention (B) under these conditions.Can from microorganism, prepare chromosomal dna library as mentioned above, described microorganism belongs to streptomyces, as abortion within the first month of pregnancy look streptomycete, brick-red streptomycete, do not produce the look streptomycete, hot lightskyblue streptomycete, the black walnut streptomycete, Tianjin island chain mould, pelletizing produces the look streptomycete, olive produces the look streptomycete, the decorative chains mould, streptomyces griseus, wool streptomycete, three damp streptomycetes, pale asphyxia streptomycete, the rose streptomycete that reddens, Shandong ground streptomycete and Regensburg streptomycete, more specifically, abortion within the first month of pregnancy look streptomycete IFO 12898, brick-red streptomycete ATCC 21469 does not produce look streptomycete IFO 12735, hot lightskyblue streptomycete IFO14273t, black walnut streptomycete IFO 13445, Tianjin island chain mould IFO 13782, pelletizing produce look streptomycete IFO 13673t, olive produces look streptomycete IFO 12444, decorative chains mould IFO 13069t, streptomyces griseus ATCC 10137, streptomyces griseus IFO 13849T, wool streptomycete IFO 12787T, three damp streptomycete IFO 13855T, pale asphyxia streptomycete IFO 13434T, the rose streptomycete IFO13682T that reddens, Shandong ground streptomycete IFO 15875T and Regensburg streptomycete IFO 13446T, etc.; Or belong to the microorganism that saccharopolyspora strain belongs to, as the Ta Shi saccharopolyspora strain, more specifically, Ta Shi saccharopolyspora strain JCM9383t etc.As can mentioning comprising SEQ ID NO:15 as the specific examples of the DNA of this probe, the DNA of nucleotide sequence shown in any one in 16,17,112,154,155,156,157,158,255,257,258,259,260,261,262,263 or 264; The DNA that comprises the partial nucleotide sequence of these nucleotide sequences; Or comprise DNA with described partial nucleotide sequence complementary nucleotide sequence.

For with host cell expression DNA of the present invention (B), for example according at " Molecular Cloning:A Laboratory Manual second edition (1989) ", Cold Spring Harbor Laboratory Press; " Current Protocols in Molecular Biology (1987) ", John Wiley ﹠amp; Conventional genetic engineering method described in the Sons, company etc. prepares DNA wherein of the present invention (B) and DNA that the promotor that works can be operatively connected, and imports in the host cell in host cell.By preparation DNA from transformant with for example carry out at " Molecular Cloning second edition " Cold Spring Harbor Press (Molecular Biology, John Wiley ﹠amp with the DNA for preparing then; Sons, the genetic engineering analytical procedure described in the N.Y. (1989) (as confirming restriction enzyme sites, dna sequencing, DNA hybridization, PCR etc.) can confirm whether the transformant that obtains comprises DNA of the present invention (B).

By will be wherein DNA of the present invention (B) and the promotor that in host cell, the works DNA that can be operatively connected import the cell that comprises DNA of the present invention (A), can in identical cell, express DNA of the present invention (B) and DNA of the present invention (A).

For example the cell that comprises DNA of the present invention (B) by cultivation can prepare protein of the present invention (B).As this cell, mention the microorganism of expressing DNA of the present invention (B) and having the ability that produces protein of the present invention (B), as from the isolating microorganism strains that comprises DNA of the present invention (B) of nature, by handling by described natural bacterial strain deutero-mutant strain with reagent or ultraviolet ray etc.For example, mention the microorganism that belongs to streptomycete, as abortion within the first month of pregnancy look streptomycete, brick-red streptomycete, do not produce the look streptomycete, light gray streptomycete, hot lightskyblue streptomycete, the black walnut streptomycete, Tianjin island chain mould, pelletizing produces the look streptomycete, and olive produces the look streptomycete, the decorative chains mould, streptomyces griseus, wool streptomycete, three damp streptomycetes, the pale asphyxia streptomycete, the rose streptomycete that reddens, Shandong ground streptomycete and Regensburg streptomycete, more specifically, abortion within the first month of pregnancy look streptomycete IFO 12898, brick-red streptomycete ATCC 21469 does not produce look streptomycete IFO 12735, light gray streptomycete ATCC 11796, hot lightskyblue streptomycete IFO 14273t, black walnut streptomycete IFO 13445, Tianjin island chain mould IFO 13782, pelletizing produce look streptomycete IFO13673t, olive produces look streptomycete IFO 12444, decorative chains mould IFO 13069t, streptomyces griseus ATCC 10137, streptomyces griseus IFO 13849T, wool streptomycete IFO 12787T, three damp streptomycete IFO 13855T, pale asphyxia streptomycete IFO 13434T, the rose streptomycete IFO 13682T that reddens, Shandong ground streptomycete IFO 15875T and Regensburg streptomycete IFO 13446T, etc.; Or belong to the saccharopolyspora strain genus, as the Ta Shi saccharopolyspora strain, more specifically, Ta Shi saccharopolyspora strain JCM 9383t etc.In addition, can mention the transformant that has wherein imported DNA of the present invention (B).Particularly for example, mention wherein will with the tac promotor, the trc promotor, the DNA of the present invention (B) that lac promotor or T7 phage promoter can be operatively connected imports colibacillary transformant.As more specific examples, mention the e. coli jm109/pKSN657FD that describes in the following embodiments, e. coli jm109/pKSN923FD, e. coli jm109/pKSN671FD etc.

Can cultivate the microorganism that comprises DNA of the present invention (B) according to the method that is generally used for culturing micro-organisms, more specifically, carry out according to the above-mentioned condition in the method for microorganism that comprises DNA of the present invention (A) in cultivation.

For example, the protein of the present invention (B) of the microorganisms producing by comprising DNA of the present invention (B) can use in the reaction system of protein of the present invention (A) by various forms, culture as the microorganism of production protein of the present invention (B), the microorganism cells of production protein of the present invention (B), by handling the material that this cell obtains, the cell-free extract of microorganism, the complete protein of purifying not, the protein of purifying etc.The above-mentioned material that obtains by the processing cell comprises for example freeze drying cell, acetone drying cell, ground cell, the autolyzate of cell, the cell of supersound process, the cell of alkaline purification, the cell that organic solvent is handled etc.Alternatively, can be adsorbed on carrier combined techniques on synthetic polymer etc. according to currently known methods such as use, be contained in comprising method and can fix protein of the present invention (B) any in the above-mentioned various forms in the polymer mesh matrix with use, be used for the reaction system of protein of the present invention (A) then.

As the method for purifying protein of the present invention (B) from the culture of the microorganism that comprises DNA of the present invention (B), can use routine to be used for method for purifying proteins.For example can mention following method.

At first,, break by physics such as supersound process then by harvested cell from the culture of microorganism such as centrifugal grade, or by using tensio-active agent or lysis enzyme such as N,O-Diacetylmuramidase chemical disruption cell.From the lysate of the generation of acquisition like this, by centrifugal, membrane filtrations etc. are removed insolubles with the preparation cell-free extract, it is then by any suitable separation and purification process, as cation-exchange chromatography, anion-exchange chromatography, hydrophobic chromatography, classifications such as gel permeation chromatography, purifying protein of the present invention (B) thus.By separating the fraction that so obtains, can be further purified protein of the present invention (B) with SDS-PAGE.

Can confirm that protein of the present invention (B) is as from ferredoxin-NADP in the reaction system of compound (I) and protein of the present invention (A) reaction as the function of ferredoxin ⁺Reductase enzyme is to the proteic function of electron transport of protein of the present invention (A).Particularly for example, by adding protein of the present invention (B) and NADPH, ferredoxin-NADP ⁺Reductase enzyme and protein of the present invention (A) are in the reaction system that compound (I) and protein of the present invention (A) react, and then detection compound (II) can confirm to the conversion of compound (III).

Control in the method for weed in the present invention, compound (I) is applied to the cultivation of plants zone of expressing protein of the present invention (A).The variant that this plant can be expressed protein of the present invention (A) maybe can be expressed a plurality of variants of protein of the present invention (A).As protein of the present invention (A), for example, can mention protein of the present invention (A).Can obtain to express the plant of protein of the present invention (A) as the transgenic plant that import DNA of the present invention (A).This importing comprises in the above described manner DNA of the present invention (A) imported vegetable cell so that DNA is in the position that its is expressed, then the transformant aftergrowth from obtaining.The DNA of the present invention (A) that imports vegetable cell can connect the nucleotide sequence of coding to the encoding transport signals of born of the same parents' inner cell organ at its upstream, so that open reading-frame (ORF) is in frame.

The plant that has wherein imported DNA of the present invention (A) and expression protein of the present invention (A) is metabolized to compound (I) compound of low weeding activity in its cell.As a result, reduce in the plant from the plant injury of herbicidal compound and give resistance described compound.Therefore, wherein imported DNA of the present invention (A) and express protein of the present invention (A) though plant also can well-grown in the situation that compound (I) is applied to its cultural area.By cultivate described plant and use above-mentioned herbicidal composition in cultural area can effectively remove except wherein imported DNA of the present invention (A) and the expression protein of the present invention (A) plant weeds.Can improve the productive rate of above-mentioned plant, improve quality, reduce the amount of used weedicide and save work.

The cell of the gene by will expressing code book invention protein (A) and described compound or described herbicidal composition contact and assess the degree of injury of pair cell, and the cell that can express protein of the present invention (A) is to formula (I) compound or comprise the resistance assessment of the herbicidal composition of described compound.

Particularly, in order to assess the microorganism cells of expressing protein of the present invention (A) resistance, can prepare the intestinal bacteria of the conversion of expressing plant PPO and protein of the present invention (A) to the herbicidal composition of compound (I) or inclusion compound (I).This preparation comprises the intestinal bacteria that in addition DNA of the present invention (A) imported the conversion that for example can be used for assessing the active inhibition of PPO and describe in Japanese patent application 11-102534, the intestinal bacteria that more specifically wherein described plant PPO genes such as U.S. Patent number 5939602 imported effectively intestinal bacteria BT3 bacterial strain and express the conversion of PPO gene.As F.Yamamoto, H.Inokuti, H.Ozaki, (1988) Japanese Journal ofGenetics, 63 volumes, the 237-249 page or leaf is described, intestinal bacteria BT3 bacterial strain defectiveness and do not have multiplication capacity in the PPO gene.By comprising the compound of 0-1.0ppm (I) or containing the about 18-24 of transformed into escherichia coli hour of producing 37 ℃ of following shaking culture in the liquid nutrient medium of herbicidal composition of described compound and can assess resistance to compound or herbicidal composition with the colibacillary propagation of the described conversion of photo densitometry of 600nm.As protein of the present invention (A), for example can mention protein of the present invention (A).

The plant of expressing protein of the present invention (A) as assessment is to formula (I) compound or comprise the method for resistance level of the herbicidal composition of described compound, mentions herbicidal composition is applied to plant and measures the plant-growth degree methods.For more quantitative confirmation, for example at first several leaves of plant are dipped in the aqueous solution of the compound (I) that comprises various concentration, perhaps the aqueous solution with compound (I) is sprayed on several pieces leaves of plant, then at room temperature is positioned on the nutrient agar in light.After a couple of days, according to Mackenney, G., J.Biol.Chem., 140; The 315th page of (1941) described method extracted chlorophyll to measure chlorophyllous content from plant leaf.Particularly for example, take the leaf of plant and be divided into 2 along the main lobe arteries and veins.Herbicidal composition is dispersed on the whole surface of one of blade.Another blade stays and is untreated.These blades are placed on the MS substratum that comprises 0.8% agar and in room temperature in light, placed 7 days.Then, in the 5ml80% aqueous acetone solution, grind each blade to extract chlorophyll with pestle and mortar.With 80% aqueous acetone solution, 10 times of extracting solutions of dilution with according to Mackenney, G., J.Biol.Chem., 140; The 315th page of (1941) described method is at 750nm, and 663nm and 645nm measure absorbancy down to calculate total chlorophyll content.The percentage point of total chlorophyll content of the blade by display process and total chlorophyll content of the blade that is untreated can comparative assessment to the degree of the resistance of compound (I).As protein of the present invention (A), for example, can mention protein of the present invention (A).

Based on the aforesaid method of assessment, can select plant or the vegetable cell of demonstration to the resistance of the herbicidal composition of compound (I) or inclusion compound (I) to the resistance level of the herbicidal composition of compound (I) or inclusion compound (I).For example, select from compound (I) or the herbicidal composition that comprises this compound are sprayed onto the plant of not seeing damage the plant growing zone, or by in the presence of compound (I), cultivating the still vegetable cell of continuous growth.Particularly for example, soil is loaded in the plastic tub with for example 10cm diameter and the 10cm degree of depth.The seed of sowing plant is also cultivated in the greenhouse.By mixing the herbicidal composition of 5 parts of inclusion compounds (I), 6 parts of Sorpols (sorpol) 3005X (Toho chemical) and 89 parts of dimethylbenzene prepare emulsion.Be dispersed in equably on all faces from the leaf of the top plant of in above-mentioned basin, cultivating with the emulsion of the dilution proportion specified quantitative of 1 hectare of 1000L and with spray gun with the water that comprises 0.1% (v/v) tackiness agent.Culturing plants is after 16 days in the greenhouse, and research is to the damage of plant.Can select wherein not observe the plant of damage or wherein damage the plant that alleviates.Further, the plant by these selections of mating can obtain progeny plants.

Embodiment

Use following examples to explain the present invention in more detail, but the invention is not restricted to these embodiment.

Carrying out the HPLC of content analysis in

embodiment

1,41 and 42 and the grading purification of compound under the condition shown below.

(HPLC analysis condition 1)

Post: SUMIPAX ODS211 (Sumika Chemical Analysis Service)

Column temperature: 35 ℃

Flow velocity: 1ml/ minute

Detect wavelength: UV 254nm

The eluent A:0.01%TFA aqueous solution

Eluent B: acetonitrile

Elution requirement: sample is expelled in the solvent mixture equilibrated post with 90% eluent A and 10% eluent B.The solvent mixture of mobile then 90% eluent A and 10% eluent B 5 minutes.The solvent mixture of this then flow eluent A and eluent B 20 minutes, the ratio with eluent B is increased to 90% from 10% simultaneously.The solvent mixture of mobile then 10% eluent A and 90% eluent B 8 minutes.

Embodiment 1 is by the metabolism of the compound (II) of microorganism

(1) metabolism of compound (II)

In ISP2 nutrient agar (1.0% (w/v) wort, 0.4% (w/v) yeast extract, 0.4% (w/v) glucose, 2.0% (w/v) agar, pH7.3) the various microorganisms shown in the middle cultivation table 1 and 2.Every kind of microorganism of loopful is added TGY substratum (0.5% (w/v) Tryptones, 0.5% (w/v) yeast extract, 0.1% (w/v) glucose, 0.01% (w/v) KH ₂PO ₄, pH7.0) and at 30 ℃ descended the vibration incubations 2-4 days.The culture that 1/10th milliliters (0.1ml) obtained comprise the 3ml sporulation meat soup of 100ppm compound (II) (0.1% (w/v) meat extract, 0.2% (w/v) tryptose, 1% glucose, pH7.1) at 30 ℃ of vibration incubations 7-8 days down.In the culture that 50 microlitres (50 μ l) 2N HCl add is produced and with the 3ml ethyl acetate extraction it.On HPLC, analyze the ethyl acetate layer that obtains.Reduce the concentration (23.9 minutes post retention time) of compound (II), the retention time 21.6 minutes and 22.2 minutes detects the new peak (respectively call oneself metabolite (I) and metabolite (II)) of compound.The result shows in table 1 and 2.

Table 1

Microorganism strains	The concentration (ppm) of compound (II)	Peak area (the x10 of metabolite (I) ⁴)	Peak area (the x10 of metabolite (II) ⁴)
Microorganism strains	The concentration (ppm) of compound (II)	Peak area (the x10 of metabolite (I) ⁴)	Peak area (the x10 of metabolite (II) ⁴)	Cocoa streptomycete A Su subspecies (Streptomyces cacaoiasoensis) IFO 13813	77.8	3.43	3.57
The brown streptomycete IFO 12870t of ash	49.5	7.96	9.86		77.8	3.43	3.57
The brown streptomycete IFO 12870t of ash	49.5	7.96	9.86	Decorative chains mould IFO 13069t	65.3	4.30	5.00
Hot lightskyblue streptomycete IFO 14273t	51.7	7.47	9.16	Decorative chains mould IFO 13069t	65.3	4.30	5.00
Hot lightskyblue streptomycete IFO 14273t	51.7	7.47	9.16	Rose produces look streptomycete (Streptomyces roseochromogenes) ATCC 13400	81.9	0.71	0.82
Lilac grey streptomycete (Streptomyces lavendulae) ATCC 11924	89.6	1.02	1.50		81.9	0.71	0.82
	89.6	1.02	1.50	Streptomyces griseus ATCC 10137	65.6	6.19	1.30
Streptomyces griseus ATCC 11429	30.3	12.8	15.6	Streptomyces griseus ATCC 10137	65.6	6.19	1.30
Streptomyces griseus ATCC 11429	30.3	12.8	15.6	Streptomyces griseus ATCC 12475	51.1	0.52	2.27
Streptomyces griseus ATCC 15395	75.2	1.91	2.26	Streptomyces griseus ATCC 12475	51.1	0.52	2.27
Streptomyces griseus ATCC 15395	75.2	1.91	2.26	Red saccharopolyspora (Streptomyces erythreus) ATCC 11635	54.6	4.94	6.05
Shot hole streptomycete (Streptomyces scabies) IFO 3111	88.3	3.28	4.40	Red saccharopolyspora (Streptomyces erythreus) ATCC 11635	54.6	4.94	6.05
Shot hole streptomycete (Streptomyces scabies) IFO 3111	88.3	3.28	4.40	Streptomyces griseus IFO 3102	22.6	14.4	18.5
Little catena mould (Streptomyces caternulae) IFO 12848	85.3	3.81	1.59	Streptomyces griseus IFO 3102	22.6	14.4	18.5
Little catena mould (Streptomyces caternulae) IFO 12848	85.3	3.81	1.59	Springtime streptomycete (Streptomyces kasugaensis) ATCC 15714	92.4	1.08	0.91
Streptomyces rimosus (Streptomyces rimosus) ATCC 10970	70.9	2.30	2.87		92.4	1.08	0.91
Streptomyces rimosus (Streptomyces rimosus) ATCC 10970	70.9	2.30	2.87	Do not produce look streptomycete IFO 12735	0.0	15.9	21.8
Streptomyces lydicus (Streptomyces lydicus) IFO 13058	62.0	5.48	6.69	Do not produce look streptomycete IFO 12735	0.0	15.9	21.8

Table 2

Microorganism strains	The concentration (ppm) of compound (II)	Peak area (the x10 of metabolite (I) ⁴)	Peak area (the x10 of metabolite (II) ⁴)
Microorganism strains	The concentration (ppm) of compound (II)	Peak area (the x10 of metabolite (I) ⁴)	Peak area (the x10 of metabolite (II) ⁴)	Abortion within the first month of pregnancy look streptomycete IFO 12898	46.4	8.28	10.5
Afghanistan streptomycete (Streptomyces afghaniensis) IFO 12831	80.6	2.54	3.59		46.4	8.28	10.5
	80.6	2.54	3.59	Streptomyces hachijoensis (Streptomyces hachijoensis) IFO 12782	83.9	4.99	2.91
Mutation (Streptomyces argenteolus var.toyonakensis) ATCC 21468 during silver sample streptomycete is rich	13.0	14.9	19.2		83.9	4.99	2.91
	13.0	14.9	19.2	Brick-red streptomycete ATCC 21469	18.4	11.6	14.4
Purple light streptomycete (Streptomyces purpurascens) ATCC 25489	70.9	5.37	6.11	Brick-red streptomycete ATCC 21469	18.4	11.6	14.4
	70.9	5.37	6.11	Ash produces look streptomycete (Streptomyces griseochromogenes) ATCC 14511	53.9	3.00	3.97
Springtime streptomycete IFO 13851	66.3	12.1	12.6		53.9	3.00	3.97
Springtime streptomycete IFO 13851	66.3	12.1	12.6	Mutation ATCC 21468t during silver sample streptomycete is rich	90.1	2.75	3.01
Rose produces look streptomycete ATCC 13400t	71.8	4.66	4.00		90.1	2.75	3.01
Rose produces look streptomycete ATCC 13400t	71.8	4.66	4.00	Black walnut streptomycete IFO 13445	12.8	21.9	24.9
Streptomyces rosechromogenus ATCC 21895	74.2	4.14	5.87	Black walnut streptomycete IFO 13445	12.8	21.9	24.9
Streptomyces rosechromogenus ATCC 21895	74.2	4.14	5.87	Streptomyces fimicarius (Streptomyces fimicarius) ATCC 21900	46.5	8.33	11.3
Streptomyces chartreusis (Streptomyces chartreusis) ATCC 21901	61.1	3.70	3.94		46.5	8.33	11.3
	61.1	3.70	3.94	Styreptomyces globispotus strain ball spore subspecies (Streptomyces globisporus subsp.globisporus) ATCC 21903	79.9	2.86	2.52
Light gray streptomycete ATCC 11796	0	14.4	19.9		79.9	2.86	2.52

Ta Shi saccharopolyspora strain JCM 9383T	82.9	5.83	7.71
Ta Shi saccharopolyspora strain JCM 9383T	82.9	5.83	7.71	Streptomyces species SANK 62585	54.6	2.30	3.44

(2) structure determination of metabolite (I) and metabolite (II)

The freezing original seed of streptomyces griseus ATCC 11429 is added the 3ml microbiological culture media, and (0.7% (w/v) gathers peptone, 0.5% (w/v) yeast extract, 1.0% (w/v) glucose, 0.5% (w/v) K ₂HPO ₄, pH7.2) and in test tube, vibrate and be incubated overnight to obtain pre-culture.This pre-culture is added in the 300ml microbiological culture media of the compound (II) that comprises 100ppm concentration.This is assigned in 100 small test tubes with every pipe 3ml, and descended the vibration incubations 6 days at 30 ℃.By adding after HCl is adjusted to pH2 with this culture of 250ml, with the 250ml ethyl acetate extraction it.From ethyl acetate layer, remove solvent.Be dissolved in resistates in the 3ml acetone and put (TLC silica-gel plate 60F to the TLC silica-gel plate ₂₅₄, 20cm x 20cm, 0.25mm is thick, Merck company).Use toluene, the 5:7:1 of formic acid and ethyl formate (v/v/v) mixture launches the TLC plate.Get the Rf value and be about 0.58 silica gel.This content with acetone extract TLC plate.From extract layer, remove acetone.Be dissolved in resistates in the 10ml acetonitrile and use the HPLC fractional separation.Recovery only comprises the fraction of metabolite (I) and metabolite (II) to obtain 3.7mg metabolite (hereinafter referred to as " metabolite A ").

Carry out the mass spectroscopy of metabolite A.Metabolite A has the quality than compound (II) little 14.In addition, analyze, determine that metabolite (A) is the compound with structure shown in the formula (III) from H-NMR.

(3) weeding activity of compound (III) test

Soil is loaded in the round plastic tub with 10cm diameter and 10cm degree of depth.Sowing and cultivation barnyard grass in the greenhouse, big fringe amur foxtail, Ivyleaf morningglory 10 days.With five (5) parts of test compounds, 6 parts of Sorpol 3005X (Toho chemical company) and 89 parts of dimethylbenzene thorough mixing are to produce emulsion.Be dispersed in equably on all faces from the leaf of the top plant of in above-mentioned basin, cultivating with the emulsion of the dilution proportion specified quantitative of 1 hectare of 1000L and with spray gun with the water that comprises 0.1% (v/v) tackiness agent.Culturing plants was studied the weeding activity of test compounds after 16 days in the greenhouse.The result represents in table 3.

Table 3

Soil is loaded in the round plastic tub with 10cm diameter and 10cm degree of depth.The sowing barnyard grass, big fringe amur foxtail, Ivyleaf momingglory.With five (5) parts of test compounds, 6 parts of Sorpol 3005X (Toho chemical company) and 89 parts of dimethylbenzene thorough mixing are to produce emulsion.With the water that comprises 0.1% (v/v) tackiness agent with the emulsion of the dilution proportion specified quantitative of 1 hectare of 1000L and be dispersed in equably with spray gun on the surface of soil.Culturing plants was studied weeding activity after 19 days in the greenhouse.The result represents in table 4.

Table 4

In the above in the table 3 and 4, the intensity of weeding activity progressively is expressed as 0,1,2,3,4,5,6,7,8,9 and 10.Rudiment or growth are used relatively and demonstration does not have the situation of difference fully or basically with being untreated during plant inspection that digital " 0 " is represented wherein to be used to test.Numeral " 10 " is represented wherein withered fully or the rudiment or the complete repressed situation of growing of plant.

The preparation of embodiment 2 protein of the present invention (A1)

(1) preparation of cell crude extract

With 100ml A substratum (0.1% (w/v) glucose in the freezing original seed adding 500ml Erlenmeyer flask of abortion within the first month of pregnancy look streptomycete IFO 12898,0.5% (w/v) Tryptones, 0.5% (w/v) yeast extract, 0.1% (w/v) dipotassium hydrogen phosphate, pH7.0) and 30 ℃ of following rotational oscillation incubations 1 day to obtain pre-culture.Add eight milliliters (8ml) pre-culture in the 200ml A substratum and under 30 ℃, shook in the bottle rotational oscillation incubation 2 days at 500ml band baffle plate.Reclaim cell precipitation by the culture centrifugal (3,000g, 5 minutes) that will produce.These cell precipitations are suspended in the 100ml B substratum (1% (w/v) glucose, 0.1% extractum carnis, 0.2% (w/v) tryptose) that comprises 100ppm compound (II) and under 30 ℃ in 500ml slope mouth flask reciprocating vibration incubation 16 hours.Reclaim cell precipitation by the culture centrifugal (3,000g, 5 minutes) that 10L is produced.The cell precipitation that produces with 1L 0.1M potassium phosphate buffer (pH7.0) washing 2 times is to provide 162g cell precipitation.

With 1g cell precipitation 2ml these cell precipitations are suspended in the 0.1M potassium phosphate buffer (pH7.0), to wherein adding 1mM PMSF, 5mM benzenyl amidine HCl, 1mM EDTA and 1mM two sulphur tritols (dithiotritol).By using French press (1000kg/cm ²) (OhtakeSeisakusho) ruptured cell obtains cell pyrolysis liquid 2 times repeatedly.After cell pyrolysis liquid is centrifugal (40,000xg, 30 minutes), reclaim supernatant liquor and 150, under the 000xg centrifugal 1 hour to reclaim supernatant liquor (hereinafter referred to as " cell crude extract ").

(2) compound (II) is converted into the mensuration of the ability of compound (III)

30 μ l reaction solutions of preparation 0.1M potassium phosphate buffer (pH7.0), it comprises 3ppm and uses ¹⁴The compound of C mark (II), 2.4mM β-NADPH (hereinafter referred to as " component A ") (OrientalYeast Company), 0.5mg/ml derive from the ferredoxin (hereinafter referred to as " B component ") (Sigma Company) of spinach, the cell crude extract that 1U/ml ferredoxin reductase (hereinafter referred to as " component C ") (Sigma Company) and 18 μ l reclaim in embodiment 2 (1).With reaction soln remain on 30 ℃ following 1 hour.In addition, prepare similarly and keep not to add be used for above-mentioned reaction soln composition, be selected from component A, the reaction soln of at least a component of B component and component C.Three microlitres (3 μ l) 2N HCl and 90 μ l ethyl acetate are added and be mixed in the every kind of reaction soln that keeps later.8, under the 000xg that the reaction soln that produces is centrifugal to reclaim 75 μ l ethyl acetate layers.After the drying under reduced pressure ethyl acetate layer, resistates is dissolved in the 6.0 μ l ethyl acetate.(5.0 μ l) puts TLC plate (TLC silica-gel plate 60F with its five microlitre ₂₅₄20cm x 20cm, 0.25mm is thick, Merck company) on.Use chloroform, the mixture of the 6:1:2 of acetate and ethyl acetate launched the TLC plate about 1 hour.Allow solvent evaporation then.The TLC plate is exposed to imaging plate (Fuji FilmCompany) to spend the night.Then, go up the analysis imaging plate at Image Analyzer BAS2000 (Fuji Film Company).Inspection is corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).The result is displayed in Table 5.

Table 5

(3) fractional separation of cell crude extract

Ammonium sulfate is added in the saturated amount of cell crude extract to 45% that obtains among the embodiment 2 (1).After stirring under the ice-cooled condition, by 12, reclaimed supernatant liquor under the 000x g in centrifugal 10 minutes.In the saturated amount of the supernatant liquor to 55% that ammonium sulfate is joined acquisition with after stirring under the ice-cooled condition, by 12, reclaimed precipitation under the 000x g in centrifugal 10 minutes.With two three propane damping fluid (pH7.0) dissolution precipitations of the 20mM of 27.5ml.This solution is carried out PD10 post (AmershamPharmacia Company) and comprises proteinic fraction (hereinafter referred to as " 45-55% ammonium sulfate fraction ") with two three propane damping fluid (pH7.0) wash-outs of 20mM to reclaim 38.5ml.

(4) separation of protein of the present invention (A1)

The 45-55% ammonium sulfate fraction that will prepare in embodiment 2 (3) is expelled in the HiLoad 26/10QSepharose HP post (Amersham Pharmacia Company).Next, after the two three propane damping fluids (pH 7.0) of the 20mM of 106ml flow to pillar, (gradient of NaCl is 0.001415M/ minute with the NaCl linear gradient, the NaCl range of concentrations is 0M-0.375M, and flow velocity is 3ml/ minute) the two three propane damping fluids of the 20mM that flows are recovered in the 25ml fraction of wash-out under the NaCl concentration of 0.21M-0.22M with classification.Further, the fraction that reclaims is carried out PD10 post (AmershamPharmacia Biotech Company) and comprise proteinic fraction with recovery with 20mM pair of three propane damping fluids (pH 7.0) wash-outs.

The fraction that reclaims is carried out PD10 post (Amersham Phamacia Biotech Company),, comprise proteinic fraction so that reclaim with buffer A (comprise the 2mM potassium phosphate buffer of 1.5mM NaCl, pH 7.0) wash-out.Next, fraction is injected among the Bio-Scale Ceramic hydroxylapatite I type pillar CHT10-I (BioRad Company).30 milliliters of (30ml) buffer A are flow to pillar.Subsequently, the buffer B of buffer A and linear gradient (the 100mM potassium phosphate buffer that comprises 0.03mMNaCl; Linear gradient increased to 50% buffer B since 100% buffer A during 100 minutes, flow velocity is 2ml/ minute) flowing together is recovered in the fraction of wash-out under the concentration of buffer B of 17%-20% with classification.Further, the fraction that reclaims is carried out PD10 post (Amersham Pharmacia Biotech Company) and comprised proteinic fraction with 0.05M potassium phosphate buffer (pH7.0) wash-out with recovery.

Use ultra-filtration membrane (Microcon YM-30, Millipore Company) that the fraction that reclaims is concentrated 20 times and be expelled in the HiLoad 16/60Superdex 75pg post (Amersham PharmaciaBiotech Company).50 mmoles (50mM) potassium phosphate buffer that comprises 0.15M NaCl (pH7.0) flows to (flow velocity 1ml/ minute) pillar.With each 2ml with the eluate classification.Will be under the elution volume of 56ml-66ml wash-out fraction separately classification reclaim.Analyze the protein that in each fraction, comprises with 10%-20%SDS-PAGE.

Be similar to embodiment 2 (2), at component A, B component, component C and usefulness ¹⁴Under the existence of the composition of C mark (II), the fraction rather than the cell crude extract in embodiment 2 (2) described reaction solns that add and keep reclaiming.To keep later reaction soln TLC to analyze to check corresponding to usefulness ¹⁴The intensity of the spot of the compound of C mark (III).Observe the protein that in above-mentioned SDS-PAGE, moves to the 47kDa position and have the fluctuation that is parallel to corresponding to the strength fluctuation of the spot of compound (III) aspect the concentration of the fraction band that adds successively.From the SDS-PAGE gel, reclaim described protein and carry out amino acid sequence analysis with protein sequencer (Applied Biosystems Company, Procise494HT, pulse liquid phase process).As a result, be provided at the aminoacid sequence of representing among the SEQ ID NO:18.In addition, after with the above-mentioned protein of tryptic digestion, at mass spectrograph (ThermoQuest Company, ion trap mass spectrometer LCQ, post: LC PackingsCompany PepMap C18 75 μ m x 150mm, solvent orange 2 A: 0.1%HOAc-H ₂O, solvent B:0.1%HOAc-methyl alcohol, gradient: begin in 30 minutes, to be increased to the linear gradient of the concentration of 100% solvent B, flow velocity: 0.2 μ l/ minute from mixture wash-out with 95% solvent orange 2 A and 5% solvent B) go up and analyze the digest that obtains.As a result, be provided at the aminoacid sequence of representing among the SEQ ID NO:19.

Embodiment 3 obtains DNA of the present invention (A1)

(1) preparation of the chromosomal DNA of abortion within the first month of pregnancy look streptomycete IFO 12898

At 50mlYEME substratum (0.3% (w/v) yeast extract, 0.5% (w/v) bactopeptone, 0.3% (w/v) wort, 1.0% (w/v) glucose, 34% (w/v) sucrose and 0.2% (v/v) 2.5MgCl ₂6H ₂O) in abortion within the first month of pregnancy look streptomycete IFO 12898 was descended the vibration incubations 1 day to 3 days at 30 ℃.Reclaim cell.With in the YEME substratum that comprises 1.4% (w/v) glycine and 60mM EDTA and the further vibration incubation 1 day of the cell suspension that obtains.From substratum, reclaim cell.With distilled water wash once after, with every 200mg cell 1ml it is resuspended in the damping fluid (100mM Tris-HCl (pH 8.0), 100mM EDTA, 10mM NaCl).The hen egg white lysozyme that adds every milliliter of two hectogamma (200 μ g/ml).Cell suspension was descended the vibration incubations 1 hour at 30 ℃.In addition, add 0.5%SDS and 1mg/ml Proteinase K.With cell suspension 55 ℃ of following incubations 3 hours.Use phenol, the mixture extraction cell suspension of chloroform and primary isoamyl alcohol 2 times is to reclaim each water layer.Next, use the mixture extraction of chloroform and primary isoamyl alcohol once to reclaim water layer.Obtain chromosomal DNA by ethanol sedimentation from water layer.

(2) preparation of the chromosomal dna library of abortion within the first month of pregnancy look streptomycete IFO 12898

With 1 unit limit enzyme Sau3AI at 37 ℃ of following 943 nanograms (943ng) chromosomal DNAs of among embodiment 3 (1), preparing of digestion 60 minutes.Separate the Digestive system that obtains with 0.7% agarose gel electrophoresis.From gel, reclaim the DNA of about 2.0kbp.According to the incidental specification sheets Prep-A-Gene of described test kit ^RDNA purification kit (Bio-Rad company) purify DNA is to obtain the solution that 10 μ l comprise target dna.With a microlitre (1 μ l) dna solution, 98ng mixes from the solution I that is connected test kit second edition (Takara Shuzo Company) with 11 μ l with the plasmid vector pUC118 that handles with dephosphorylation with restriction enzyme BmHI digestion and is incubated overnight under 16 ℃.Use 5 μ l to connect solution transformed into escherichia coli DH5 α.Intestinal bacteria are spent the night 30 ℃ of following shaking culture.From the substratum that obtains, reclaim intestinal bacteria.Extract plasmid so that chromosomal dna library to be provided.

(3) separation of DNA of the present invention (A1)

Carry out PCR (Fig. 1) by the chromosomal DNA that will in embodiment 3 (1), prepare as template.As primer, use oligonucleotide with nucleotide sequence shown in the SEQ ID NO:35 and pairing (hereinafter referred to as " primer is to 1 ") with oligonucleotide of nucleotide sequence shown in the SEQ ID NO:36.Based on nucleotide sequence shown in the nucleotide sequence design SEQ IDNO:35 of aminoacid sequence shown in the coding SEQ ID NO:18.In addition, design nucleotide sequence shown in the SEQ ID NO:36 based on nucleotide sequence complementary nucleotide sequence with aminoacid sequence shown in the coding SEQ ID NO:19.By adding 2 kinds every kind primer that amounts to 200nM, the above-mentioned chromosomal DNA of 250ng, the 0.5 μ ldNTP mixture (mixture of every kind of 10mM of 4 kinds of dNTP; Clontech company), 5 μ l 5xGC genome PCR reaction buffers (Clontech company), 1.1 μ l25mMMg (OAc) ₂, 5 μ l 5MGC-Melt (Clontech company) and 0.5 μ l Advantage-GC genome polysaccharase mixture (Clontech company) and distilled water, the PCR reaction soln amounts to 25 μ l.The reaction conditions of PCR is to remain on 95 ℃ after 1 minute, repeating 30 circulations, circulation comprise remain on 94 ℃ 15 seconds, then 60 ℃ 30 seconds, then 72 ℃ 1 minute, remain on then 72 ℃ 5 minutes.After maintenance, reaction soln is carried out 4% agarose gel electrophoresis.Recovery comprises the gel area of the DNA of about 150bp.By use the QIA PhastGel to extract test kit (Qiagen company) purify DNA from the gel that reclaims according to incidental specification sheets.According to the incidental specification sheets of described carrier the DNA that obtains is connected to TA cloning vector pCR2.1 (Invitrogen company) and imports intestinal bacteria TOP10F '.Use QIAprep Spin Miniprep test kit (Qiagen company) from the intestinal bacteria transformant that obtains, to prepare plasmid DNA.According to the incidental specification sheets of described test kit, use-21M13 primer (Applied Biosystems Japanese firm) and M13Rev primer (AppliedBiosystems Japanese firm) stop cycle sequencing FS easy reaction test kit (Applied Biosystems Japanese firm) with dyestuff and carry out sequencing reaction as primer.Sequencing reaction uses the plasmid DNA that obtains as template.With dna sequencing instrument 373A (Applied Biosystems Japanese firm) analytical reaction product.As a result, be provided at the nucleotide sequence shown in the Nucleotide 36 to 132 of the nucleotide sequence of representing among the SEQ ID NO:9.The aminoacid sequence of described nucleotide sequence coded expression in the amino acid/11 2 to 23 of aminoacid sequence shown in the SEQ ID NO:18.In this, expect the part of described dna encoding protein of the present invention (A1).

Next, above similar with Advantage-GC genome polysaccharase mixture (Clontech company) be used as template by the chromosomal DNA that will in embodiment 3 (2), prepare and carry out PCR.Use have nucleotide sequence shown in the SEQ ID NO:37 oligonucleotide and have the pairing (hereinafter referred to as " primer is to 2 ") of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:38 or have nucleotide sequence shown in the SEQID NO:39 oligonucleotide and have the pairing (hereinafter referred to as " primer is to 3 ") of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:40 as primer.

Next, has the wherein DNA of the nucleotide sequence of Nucleotide shown in the Nucleotide 132 of nucleotide sequence shown in 3 ' the terminal extend through SEQ ID NO:9 by pcr amplification.By using primer that 2 reaction solns that obtain are carried out PCR as template solution with by the pairing (hereinafter referred to as " primer is to 4 ") that use has the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:41 and has an oligonucleotide of nucleotide sequence shown in the SEQ ID NO:38 as primer.Similarly, has the wherein DNA of the nucleotide sequence of Nucleotide shown in the Nucleotide 36 of nucleotide sequence shown in 5 ' the terminal extend through SEQ ID NO:9 by pcr amplification.By using primer that 3 reaction solns that obtain are carried out PCR as template solution with by the pairing (hereinafter referred to as " primer is to 5 ") that use has the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:42 and has an oligonucleotide of nucleotide sequence shown in the SEQ ID NO:40 as primer.Above being similar to, with use primer to the 2kb DNA of 4 amplifications and use primer to the 150bp dna clones of 5 amplifications in TA cloning vector pCR2.1.Use QIAprep Spin Miniprep test kit (Qiagen company) from the intestinal bacteria transformant that obtains, to prepare plasmid DNA.According to the incidental specification sheets of described test kit, oligonucleotide shown in use-21M13 primer (AppliedBiosystems Japanese firm) and M13Rev primer (Applied Biosystems Japanese firm) and the SEQ ID NO:43-50 stops cycle sequencing FS easy reaction test kit (Applied Biosystems Japanese firm) with dyestuff and carries out sequencing reaction as primer.Sequencing reaction uses the plasmid DNA that obtains as template.With dna sequencing instrument 373A (Applied Biosystems Japanese firm) analytical reaction product.As will be, be provided at represented nucleotide sequence in the Nucleotide 133 to 1439 of nucleotide sequence shown in the SEQ ID NO:9 by using the result of primer to the nucleotide sequence order-checking of the 2kbp DNA of 4 amplifications.In addition, as will be, be provided at represented nucleotide sequence in the Nucleotide 1 to 35 of nucleotide sequence shown in the SEQ ID NO:9 by using the result of primer to the nucleotide sequence order-checking of the 150bp DNA of 5 amplifications.As the result who connects the nucleotide sequence that obtains, obtain the nucleotide sequence of in SEQ ID NO:9, representing.In described nucleotide sequence, there are two open reading-frame (ORF)s (ORF).Therefore, comprise by 1227 Nucleotide (comprising terminator codon) and form and the nucleotide sequence (SEQ ID NO:6) of 408 amino-acid residues of encoding and form and the nucleotide sequence (SEQ ID NO:15) of 66 amino-acid residues of encoding by 201 Nucleotide (comprising terminator codon).The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:6 (SEQ ID NO:1) it is calculated that and is 45213Da.In addition, comprise aminoacid sequence of measuring from the N-terminal amino acid sequencing of protein of the present invention (A1) (SEQ ID NO:18) and the aminoacid sequence of measuring from the segmental amino acid sequencing of tryptic digestion with mass spectroscopy (SEQ ID NO:19) by described nucleotide sequence coded aminoacid sequence.The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQID NO:15 (SEQ ID NO:12) it is calculated that and is 6818Da.

Embodiment 4 expression of protein of the present invention (A1) in intestinal bacteria

(1) contains the generation of the transformed into escherichia coli of protein of the present invention (A1)

By will in embodiment 3 (1), carrying out PCR as template with by use Expand High Fidelity PCR System (RocheMolecular Biochemicals Company) by the chromosomal DNA from abortion within the first month of pregnancy look streptomycete IFO 12898 preparations.As primer, use oligonucleotide and have the pairing (hereinafter referred to as " primer is to 19 ") of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:52 or have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:51 and have the pairing (hereinafter referred to as " primer is to 20 ") of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:53 with nucleotide sequence shown in the SEQ IDNO:51.By adding 2 kinds every kind primer that amounts to 300nM, the above-mentioned chromosomal DNA of 50ng, 5.0 μ l dNTP mixtures (mixture of every kind of 2.0mM of 4 kinds of dNTP), 5.0 μ l 10x Expand HF damping fluids (comprise MgCl ₂) and 0.75 μ l Expand HiFi enzyme mixture and distilled water, the PCR reaction soln amounts to 50 μ l.The reaction conditions of PCR is to remain on 97 ℃ after 2 minutes; Repeat 10 circulations, circulation comprise remain on 97 ℃ 15 seconds, then 65 ℃ 30 seconds, then 72 ℃ 2 minutes; Carry out 15 circulations then, circulation comprise remain on 97 ℃ 15 seconds, then 68 ℃ 30 seconds, then 72 ℃ 2 minutes (wherein each circulation increase by 20 seconds remain on 72 ℃); Remain on then 72 ℃ 7 minutes.After maintenance, reaction soln is carried out 1% agarose gel electrophoresis.Use the gel area that reclaims the DNA that comprises about 1.2kbp the gel of primer to 19 reaction soln from experience.Use the gel area that reclaims the DNA that comprises about 1.5kbp the gel of primer to 20 reaction soln from experience.By using the QIA PhastGel to extract test kit (Qiagen company) purify DNA from the gel of each recovery according to incidental specification sheets.According to the incidental specification sheets of described carrier the DNA that obtains is connected to TA cloning vector pCR2.1 (Invitrogen company) and imports intestinal bacteria TOP10F '.Use QIAprep Spin Miniprep test kit (Qiagen company) from the intestinal bacteria transformant that obtains, to prepare plasmid DNA.According to the incidental specification sheets of described test kit, use as primer-21M13 primer (AppliedBiosystems Japanese firm) and M13Rev primer (Applied Biosystems Japanese firm), have the oligonucleotide and oligonucleotide of nucleotide sequence shown in the SEQ ID NO:43, stop cycle sequencing FS easy reaction test kit (Applied Biosystems Japanese firm) with dyestuff and carry out sequencing reaction with nucleotide sequence shown in the SEQ ID NO:46.Sequencing reaction uses the plasmid DNA that obtains as template.With dna sequencing instrument 373A (Applied Biosystems Japanese firm) analytical reaction product.Based on the result, will contain the plasmid called after pCR657 of nucleotide sequence shown in the SEQ ID NO:6 and will contain the plasmid called after pCR657F of nucleotide sequence shown in the SEQ ID NO:9.

In addition, the oligonucleotide that will have nucleotide sequence shown in the SEQ ID NO:134 is annealed so that joint (Figure 47) to be provided together with the oligonucleotide with nucleotide sequence shown in the SEQID NO:135.With HindIII and XmnI digested plasmid pKSN24R2 (Akiyoshi-ShibaTa M. etc., Eur.J.Biochem.224:P335 (1994)).Joint is inserted the DNA of the about 3kb that obtains.With the plasmid called after pKSN2 (Fig. 4) that obtains.

Next, each among usefulness restriction enzyme NdeI and HindIII digestion pCR657 and the pCR657F.Digestion product is carried out agarose gel electrophoresis.Cutting comprises the gel area of about 1.2kbp DNA from the gel that carries out the pCR657 digestion product.Cutting comprises the gel area of about 1.5kbp DNA from the gel that carries out the pCR657F digestion product.By using the QIA PhastGel to extract test kit (Qiagen company) purify DNA from the gel of each recovery according to incidental specification sheets.According to shown in the incidental specification sheets of test kit connect the DNA and the plasmid pKSN2 of the every kind of acquisition that digests with NdeI and HindIII and import e. coli jm109 with connecting test kit version 1 (Takara Shuzo Company).Prepare plasmid DNA from the intestinal bacteria transformant that obtains.Analyze its structure.Will be wherein between the NdeI site and HindIII site of about 1.2kbp DNA insertion pKSN2 of code book invention protein (A1), comprise the plasmid called after pKSN657 of nucleotide sequence shown in the SEQ ID NO:6.In addition, will be wherein between the NdeI site and HindIII site of about 1.5kbp DNA insertion pKSN2 of code book invention protein (A1), comprise the plasmid called after pKSN657F of nucleotide sequence shown in the SEQ ID NO:9.With each the importing e. coli jm109 among above-mentioned plasmid pKSN657 and the pKSN657F.With intestinal bacteria transformant difference called after JM109/pKSN657 and the JM109/pKSN657F that obtains.In addition, plasmid pKSN2 is imported e. coli jm109.The intestinal bacteria transformant called after JM109/pKSN2 that obtains.

(2) expression and the described recovery of protein of protein of the present invention (A1) in intestinal bacteria

With e. coli jm109/pKSN657, under each comfortable 37 ℃ of JM109/pKSN657F and the JM109/pKSN2 at the 10ml TB substratum that comprises 50 μ g/ml penbritins (1.2% (w/v) Tryptones, 2.4% (w/v) yeast extract, 0.4% (w/v) glycerine, the 17mM potassium primary phosphate, the 72mM dipotassium hydrogen phosphate) middle overnight incubation.The culture that one milliliter (1ml) obtained is transferred in the 100rml TB substratum that comprises 50 μ g/ml penbritins and cultivation under 26 ℃.When OD660 arrives approximately 0.5 the time, the 5-amino-laevulic acid is added to the final concentration of 500 μ M, continue to cultivate.After 30 (30) minutes,, further cultivated 17 hours the final concentration that IPTG adds to 1mM.

From each substratum, reclaim cell, wash and be suspended in the above-mentioned damping fluid that 10ml comprises 1mMPMSF with 0.1Mtris-HCl damping fluid (pH7.5).Is 3 with the cell suspension that obtains in work output, carries out ultrasonoscope (Sonifer (Branson Sonic Power Company)) under the condition of space factor (dutycycle) 30% 6 times, each 3 minutes, so that obtain cell pyrolysis liquid.With cell pyrolysis liquid centrifugal (1,200xg, 5 minutes) back recovery supernatant liquor and centrifugal (150,000xg, 70 minutes) with reclaim the supernatant liquor fraction (below, the supernatant liquor fraction that will obtain from e. coli jm109/pKSN657 is called " intestinal bacteria pKSN657 extract ", the supernatant liquor fraction that will obtain from e. coli jm109/pKSN657F is called " intestinal bacteria pKSN657F extract ", and the supernatant liquor fraction that will obtain from e. coli jm109/pKSN2 is called " intestinal bacteria pKSN2 extract ").On 15%-25%SDS-PAGE, analyze the above-mentioned supernatant liquor fraction of a microlitre (1 μ l) and use Coomassie blue (hereinafter referred to as " CBB ") dyeing.As a result, all detect in intestinal bacteria pKSN657 extract and intestinal bacteria pKSN657F extract than intestinal bacteria pKSN2 extract intensive band significantly more, it is positioned at the electrophoresis position corresponding to the 47kDa molecular weight.In intestinal bacteria pKSN657F extract, detect than intestinal bacteria pKSN657 extract intensive band more.Show that e. coli jm109/pKSN657F expresses protein of the present invention (A1) than e. coli jm109/pKSN657 higher degree ground.

(3) compound (II) is converted into the detection of the ability of compound (III)

Prepare the reaction soln of 30 μ l and remain on 30 ℃ following 1 hour.Reaction soln is made up of 0.1M potassium phosphate buffer (pH7.0), and it comprises 3ppm and uses ¹⁴The compound of C mark (II), 2mM β-NADPH (hereinafter referred to as " component A ") (Oriental Yeast Company), 0.2mg/ml derive from the ferredoxin (hereinafter referred to as " B component ") (Sigma Company) of spinach, the supernatant liquor fraction that 1U/ml ferredoxin reductase (hereinafter referred to as " component C ") (Sigma Company) and 18 μ l reclaim in embodiment 4 (2).In addition, prepare similarly and keep not to add be used for above-mentioned reaction soln composition, be selected from component A, the reaction soln of at least a component of B component and component C.Three microlitres (3 μ l) 2N HCl and 90 μ, 1 ethyl acetate are added and be stirred in the every kind of reaction soln that keeps later.8, under the 000xg that the reaction soln that produces is centrifugal to reclaim 75 μ l ethyl acetate layers.After the drying under reduced pressure ethyl acetate layer, resistates is dissolved in the 6.0 μ l ethyl acetate.(5.0 μ l) puts TLC plate (TLC silica-gel plate 60F with its five microlitre ₂₅₄20cm x 20cm, 0.25 is thick, Merck company) on.Use chloroform, the mixture of the 6:1:2 of acetate and ethyl acetate launched the TLC plate about 1 hour.Allow solvent evaporation then.The TLC plate is exposed to imaging plate (FujiFilm Company) to spend the night.Then, go up the analysis imaging plate at Image Analyzer BAS2000 (Fuji FilmCompany).Inspection is corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).The result is displayed in Table 6.

Table 6

The preparation of embodiment 5 protein of the present invention (A2)

(1) preparation of cell crude extract

With 10ml A substratum (0.1% (w/v) glucose in the freezing original seed adding 10ml test tube of Ta Shi saccharopolyspora strain JCM9383t, 0.5% (w/v) Tryptones, 0.5% (w/v) yeast extract, 0.1% (w/v) dipotassium hydrogen phosphate, pH 7.0) and 30 ℃ down vibration incubations 1 day to obtain pre-culture.Add eight milliliters (8ml) pre-culture in the 200ml A substratum and under 30 ℃, shook in the bottle rotating and culturing 2 days at 500ml band baffle plate.Reclaim cell precipitation by the culture centrifugal (3,000g, 10 minutes) that 10L is produced.These cell precipitations are suspended in the 100ml B substratum (1% (w/v) glucose, 0.1% extractum carnis, 0.2% (w/v) tryptose) that comprises 100ppm compound (II) and under 30 ℃ in 500ml slope mouth flask reciprocating vibration incubation 20 hours.Reclaim cell precipitation by the culture centrifugal (3,000xg, 10 minutes) that 10L is produced.The cell precipitation that produces with 1L0.1M potassium phosphate buffer (pH 7.0) washing 2 times is to provide 119g cell precipitation.

With 1g cell precipitation 2ml these cell precipitations are suspended in the 0.1M potassium phosphate buffer (pH7.0).Add a mmole (1mM) PMSF, 5mM benzenyl amidine HCl, 1mM EDTA, 3 μ g/ml leupeptins, 3 μ g/ml pepstatins and 1mM two sulphur tritols.By using French press (1000kg/cm ²) (Ohtake Seisakusho) break repeatedly suspension 2 times obtains cell pyrolysis liquid.After cell pyrolysis liquid is centrifugal (40,000xg, 30 minutes), reclaim supernatant liquor and 150, under the 000xg centrifugal 1 hour to reclaim supernatant liquor (hereinafter referred to as " cell crude extract ").

30 μ l reaction solns of preparation 0.1M potassium phosphate buffer (pH 7.0), it comprises 3ppm and uses ¹⁴The compound of C mark (II), 2.4mM β-NADPH (hereinafter referred to as " component A ") (OrientalYeast Company), 0.5mg/ml derive from the ferredoxin (hereinafter referred to as " B component ") (Sigma Company) of spinach, the cell crude extract that 1U/ml ferredoxin reductase (hereinafter referred to as " component C ") (Sigma Company) and 18 μ l reclaim in embodiment 5 (1).With reaction system remain on 30 ℃ following 1 hour.In addition, prepare similarly and keep not to add be used for above-mentioned reaction soln composition, be selected from component A, the reaction soln of at least a component of B component and component C.Three microlitres (3 μ l) 2N HCl and 90 μ l ethyl acetate are added and be mixed in the every kind of reaction soln that keeps later.8, under the 000xg that the reaction soln that produces is centrifugal to reclaim 75 μ l ethyl acetate layers.After the drying under reduced pressure ethyl acetate layer, resistates is dissolved in the 6.0 μ l ethyl acetate.(5.0 μ l) puts TLC plate (TLC silica-gel plate 60F with its five microlitre ₂₅₄20cm x 20cm, 0.25 is thick, Merck company) on.Use chloroform, the mixture of the 6:1:2 of acetate and ethyl acetate launched the TLC plate about 1 hour.Allow solvent evaporation then.The TLC plate is exposed to imaging plate (Fuji FilmCompany) to spend the night.Then, go up the analysis imaging plate at Image Analyzer BAS2000 (Fuji Film Company).Inspection is corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).The result is displayed in Table 7.

Table 7

(3) fractional separation of cell crude extract

Ammonium sulfate is added in the saturated amount of cell crude extract to 45% that obtains among the embodiment 5 (1).Under ice-cooled condition, stir, by 12, reclaimed supernatant liquor under the 000xg in centrifugal 10 minutes.In the saturated amount of the supernatant liquor to 55% that ammonium sulfate add is obtained with after stirring under the ice-cooled condition, by 12, reclaimed precipitation under the 000xg in centrifugal 10 minutes.With two three propane damping fluid (pH7.0) dissolution precipitations of the 20mM of 32.5ml.This solution is carried out PD10 post (AmershamPharmacia Company) and comprises proteinic fraction (hereinafter referred to as " 45-55% ammonium sulfate fraction ") with two three propane damping fluids (pH 7.0) wash-outs of 20mM to reclaim 45.5ml.

(4) separation of protein of the present invention (A2)

The 45-55% ammonium sulfate fraction that will prepare in embodiment 5 (3) is expelled in the HiLoad26/10QSepharose HP post (Amersham Pharmacia Company).Next, after the two three propane damping fluids (pH7.0) of the 20mM of 100ml flow to pillar, (gradient of NaCl is 0.004M/ minute with the NaCl linear gradient, the NaCl range of concentrations is 0M-0.5M, and flow velocity is 8ml/ minute) the two three propane damping fluids of the 20mM that flows are recovered in the 30ml fraction of wash-out under the NaCl concentration of 0.25M-0.26M with classification.Further, the fraction that reclaims is carried out PD10 post (AmershamPharmacia Biotech Company) and comprise proteinic fraction with recovery with 20mM pair of three propane damping fluid (pH7.0) wash-outs.

The fraction that reclaims is carried out PD10 post (Amersham Pharmacia Biotech Company), and (the 2mM potassium phosphate buffer that comprises 1.5mM NaCl, pH7.0) wash-out comprise proteinic fraction so that reclaim with buffer A.Next, fraction is injected among the Bio-Scale Ceramic hydroxylapatite I type pillar CHT10-I (BioRad Company).20 milliliters of (20ml) buffer A are flow to pillar.Subsequently, the buffer B of buffer A and linear gradient (the 100mM potassium phosphate buffer that comprises 0.03mMNaCl; Linear gradient increased to 50% buffer B since 100% buffer A during 100 minutes, flow velocity is 2ml/ minute) flow together and return the fraction that 10ml is received in wash-out under the concentration of buffer B of 23%-25% with classification.Further, the fraction that reclaims is carried out PD10 post (Amersham Pharmacia Biotech Company) and comprised proteinic fraction with 0.05M potassium phosphate buffer (pH7.0) wash-out with recovery.

Use ultra-filtration membrane (Microcon YM-30, Millipore Company) is concentrated into about 770 μ l with the fraction that reclaims and is expelled in the HiLoad 16/60 Superdex 75pg post (AmershamPharmacia Biotech Company).50 mmoles (50mM) potassium phosphate buffer that comprises 0.15M NaCl (pH 7.0) flows to (flow velocity 1ml/ minute) pillar.With each 2ml with the elutriant classification.Will be under the elution volume about 61ml wash-out fraction separately classification reclaim.Analyze the protein that in each fraction, comprises with 10%-20%SDS-PAGE.

Be similar to embodiment 5 (2), at component A, B component, component C and usefulness ¹⁴Under the existence of the compound of C mark (II), the fraction rather than the cell crude extract in embodiment 5 (2) described reaction solns that add and keep reclaiming.To keep later reaction soln TLC to analyze to check corresponding to usefulness ¹⁴The intensity of the spot of the compound of C mark (III).Observe the protein that in above-mentioned SDS-PAGE, moves to the 47kDa position and have the fluctuation that is parallel to corresponding to the strength fluctuation of the spot of compound (III) aspect the concentration of the fraction band that adds successively.From the SDS-PAGE gel, reclaim described protein and carry out amino acid sequence analysis with order-checking N-terminal aminoacid sequence with protein sequencer (Applied Biosystems Company, Procise494HT, pulse liquid phase process).As a result, be provided at the aminoacid sequence of representing among the SEQ ID NO:20.In addition, after with the above-mentioned protein of tryptic digestion, at mass spectrograph (ThermoQuest Company, ion trap mass spectrometer LCQ, post: LC Packings Company PepMap C18 75 μ m x 150mm, solvent orange 2 A: 0.1%HOAc-H ₂O, solvent B:0.1%HOAc-methyl alcohol, gradient: begin in 30 minutes, to be increased to the linear gradient of the concentration of 100% solvent B, flow velocity: 0.2 μ l/ minute from mixture wash-out with 95% solvent orange 2 A and 5% solvent B) go up and analyze the digest that obtains.As a result, be provided at the aminoacid sequence of representing among the SEQ IDNO:21.

Embodiment 6 obtains DNA of the present invention (A2)

(1) preparation of the chromosomal DNA of Ta Shi saccharopolyspora strain JCM 9383t

At 50mlYEME substratum (0.3% (w/v) yeast extract, 0.5% (w/v) bactopeptone, 0.3% (w/v) wort, 1.0% (w/v) glucose, 34% (w/v) sucrose and 0.2% (v/v) 2.5MgCl ₂6H ₂O) in Ta Shi saccharopolyspora strain JCM 9383t was descended the vibration incubations 1 day to 3 days at 30 ℃.Reclaim cell.With in the YEME substratum that comprises 1.4% (w/v) glycine and 60mMEDTA and the further vibration incubation 1 day of the cell suspension that obtains.From substratum, reclaim cell.With distilled water wash once after, with every 200mg cell precipitation 1ml with it be resuspended in damping fluid (100mM Tris-HCl (pH 8.0), 100mM EDTA, 10mMNaCl) in.The hen egg white lysozyme that adds every milliliter of two hectogamma (200 μ g/ml).Cell suspension was descended the vibration incubations 1 hour at 30 ℃.In addition, add 0.5%SDS and 1mg/ml Proteinase K.With cell suspension 55 ℃ of following incubations 3 hours.With phenol chloroform isoamyl alcohol extraction cell suspension 2 times to reclaim each water layer.Next, extract once to reclaim water layer with chloroform isoamyl alcohol.Obtain chromosomal DNA by the ethanol sedimentation water layer.

(2) preparation of the chromosomal dna library of Ta Shi saccharopolyspora strain JCM 9383t

With 0.78U restriction enzyme Sau3AI at 37 ℃ of following 19 micrograms (19 μ g) chromosomal DNAs of among embodiment 5 (1), preparing of digestion 60 minutes.Separate the Digestive system that obtains with 1% agarose gel electrophoresis.From gel, reclaim the DNA of about 2.0kbp.Extract test kit (Qiagen company) purify DNA and concentrated to obtain the solution that 10 μ l comprise target dna according to the incidental specification sheets of described test kit with the QIA PhastGel with ethanol sedimentation.With eight microlitres (8 μ l) dna solution, 100ng mixes from the solution I that is connected test kit second edition (Takara Shuzo Company) with 12 μ l with the plasmid vector pUC118 that handles with dephosphorylation with restriction enzyme BamHI digestion and kept 3 hours down at 16 ℃.With connecting solution transformed into escherichia coli DH5 α.With intestinal bacteria transformant overnight incubation in the LB nutrient agar that is comprising the 50mg/l penbritin under 37 ℃.From nutrient agar, reclaim the bacterium colony that obtains.Extract plasmid and be called chromosomal dna library.

(3) separation of DNA of the present invention (A2)

Carry out PCR (Fig. 2) as template with Expand HiFiPCR system (Boehringer Manheim Company) by the chromosomal DNA that will in embodiment 6 (1), prepare.As primer, use oligonucleotide with nucleotide sequence shown in the SEQ ID NO:54 and pairing (hereinafter referred to as " primer is to 6 ") with oligonucleotide of nucleotide sequence shown in the SEQ ID NO:55.Based on nucleotide sequence shown in the nucleotide sequence design SEQ ID NO:54 of N-terminal aminoacid sequence shown in the coding SEQ ID NO:20.In addition, design nucleotide sequence shown in the SEQ ID NO:55 based on nucleotide sequence complementary nucleotide sequence with internal amino acid sequence shown in the coding SEQ ID NO:21.By the above-mentioned chromosomal DNA of adding 300ng, 2 kinds every kind primer that amounts to 7.5pmol, 0.2 μ ldNTP mixture (mixture of every kind of 2mM of 4 kinds of dNTP), 2.5 μ l 10x damping fluids (comprise MgCl ₂), 0.19 μ l Expand HiFi enzyme mixture and distilled water, the PCR reaction soln amounts to 25 μ l.The reaction conditions of PCR is to remain on 97 ℃ after 2 minutes, repeating 10 circulations, circulation comprise remain on 97 ℃ 15 seconds, then 65 ℃ 30 seconds, then 72 ℃ 1 minute; Carry out 15 circulations then, circulation comprise remain on 97 ℃ 15 seconds, then 65 ℃ 30 seconds, then 72 ℃ 1 minute (wherein each circulation increase remain on 72 ℃ following 20 seconds); Remain on then 72 ℃ 7 minutes.After maintenance, reaction soln is carried out 2% agarose gel electrophoresis.Recovery comprises the gel area of the DNA of about 800bp.By use the Qiagen PhastGel to extract test kit (Qiagen company) purify DNA from the gel that reclaims according to incidental specification sheets.According to the incidental specification sheets of described carrier the DNA that obtains is connected to TA cloning vector pCRII-TOPO (Invitrogen company) and imports intestinal bacteria TOP10F '.Use Qiagen Tip20 (Qiagen company) from the intestinal bacteria transformant that obtains, to prepare plasmid DNA.According to the incidental specification sheets of described test kit, use-21M13 primer (Applied Biosystems Japanese firm) and M13Rev primer (Applied Biosystems Japanese firm) stop cycle sequencing FS easy reaction test kit (AppliedBiosystems Japanese firm) with dyestuff and carry out sequencing reaction as primer.With dna sequencing instrument 373A (AppliedBiosystems Japanese firm) analytical reaction product.As a result, be provided at the nucleotide sequence shown in the Nucleotide 36 to 819 of the nucleotide sequence of representing among the SEQ ID NO:10.Aminoacid sequence shown in the Nucleotide 37-60 encoding part SEQ ID NO:20 of nucleotide sequence shown in the SEQ ID NO:10.In this, expect the part of described dna encoding protein of the present invention (A2).

Next, by the chromosomal DNA that will in embodiment 6 (2), prepare as template and similar above carry out PCR with Expand HiFi PCR system.Use has the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:56 and has the pairing (hereinafter referred to as " primer is to 7 ") of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:57 as primer.By using these primers to carry out PCR, amplification has the wherein DNA of the nucleotide sequence of Nucleotide shown in the Nucleotide 36 of nucleotide sequence shown in 5 ' the terminal extend through SEQ ID NO:10.In addition, use has the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:58 and has the pairing (hereinafter referred to as " primer is to 8 ") of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:59 as primer.By using these primers to carry out PCR, amplification has the wherein DNA of the nucleotide sequence of Nucleotide shown in the Nucleotide 819 of nucleotide sequence shown in 3 ' the terminal extend through SEQ ID NO:10.Each is cloned among the TA cloning vector pCRII-TOPO to the 8 0.4kb DNA that increase with using primer to the 7 1.3kb DNA that increase with using primer.Use Qiagen Tip 20 (Qiagen companies) from the intestinal bacteria transformant that obtains, to prepare plasmid DNA.According to the incidental specification sheets of described test kit, oligonucleotide shown in use-21M13 primer (AppliedBiosystems Japanese firm) and M13Rev primer (Applied Biosystems Japanese firm) and the SEQ ID NO:60 stops cycle sequencing FS easy reaction test kit (Applied Biosystems Japanese firm) with dyestuff and carries out sequencing reaction as primer.With dna sequencing instrument 373A (Applied Biosystems Japanese firm) analytical reaction product.As will be, be provided at represented nucleotide sequence in the Nucleotide 1 to 35 of nucleotide sequence shown in the SEQ ID NO:10 by using the result of primer to the nucleotide sequence order-checking of the 1.3kb DNA of 7 amplifications.In addition, as will be, be provided at represented nucleotide sequence in the Nucleotide 819 to 1415 of nucleotide sequence shown in the SEQ IDNO:10 by using the result of primer to the nucleotide sequence order-checking of the 0.4kb DNA of 8 amplifications.As the result who connects the nucleotide sequence that obtains, obtain the nucleotide sequence of in SEQ ID NO:10, representing.In described nucleotide sequence, there are two open reading-frame (ORF)s (ORF).Therefore, comprise by 1206 Nucleotide (comprising terminator codon) and form and the nucleotide sequence (SEQ ID NO:7) of 401 amino-acid residues of encoding and form and the nucleotide sequence (SEQ ID NO:16) of 65 amino-acid residues of encoding by 198 Nucleotide (comprising terminator codon).The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:7 (SEQ ID NO:2) it is calculated that and is 43983Da.In addition, comprise aminoacid sequence of measuring from the N-terminal amino acid sequencing of protein of the present invention (A2) (SEQ ID NO:20) and the aminoacid sequence of measuring from the segmental amino acid sequencing of tryptic digestion with mass spectroscopy (SEQ IDNO:21) by described nucleotide sequence coded aminoacid sequence.The proteinic molecular weight of being made up of aminoacid sequence (SEQ IDNO:13) nucleotide sequence coded shown in the SEQ ID NO:16 it is calculated that and is 6707Da.

Embodiment 7 expression of protein of the present invention (A1) in intestinal bacteria

(1) contains the generation of the transformed into escherichia coli of protein of the present invention (A2)

By will in embodiment 6 (1), carrying out PCR as template with by use Expand HiFi PCR System (Boehringer ManheimCompany) by the chromosomal DNA from Ta Shi saccharopolyspora strain JCM 9383t preparation.As primer, use oligonucleotide and have the pairing (hereinafter referred to as " primer is to 21 ") of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:62 or have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:61 and have the pairing (hereinafter referred to as " primer is to 22 ") of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:63 with nucleotide sequence shown in the SEQ ID NO:61.By adding 2 kinds every kind primer that amounts to 300nM, the above-mentioned chromosomal DNA of 50ng, 5.0 μ l dNTP mixtures (mixture of every kind of 2.0mM of 4 kinds of dNTP), 5.0 μ l10xExpandHF damping fluids (comprise MgCl ₂) and 0.75 μ l Expand HiFi enzyme mixture and distilled water, the PCR reaction soln amounts to 50 μ l.The reaction conditions of PCR is to remain on 97 ℃ after 2 minutes; Repeat 10 circulations, circulation comprise remain on 97 ℃ 15 seconds, then 60 ℃ 30 seconds, then 72 ℃ 1 minute; Carry out 15 circulations then, circulation comprise remain on 97 ℃ 15 seconds, then 60 ℃ 30 seconds, then 72 ℃ 1 minute (wherein each circulation increase by 20 seconds remain on 72 ℃); Remain on then 72 ℃ 7 minutes.After maintenance, reaction soln is carried out 1% agarose gel electrophoresis.Utilize primer to reclaiming the gel area of the DNA that comprises about 1.2kbp the gel of 21 reaction soln from experience.Utilize primer to reclaiming the gel area of the DNA that comprises about 1.4kbp the gel of 22 reaction soln from experience.By using the Qiagen PhastGel to extract test kit (Qiagen company) purify DNA from the gel of each recovery according to incidental specification sheets.According to the incidental specification sheets of described carrier the DNA that obtains is connected to cloning vector pCRII-TOPO (Invitrogen company) and imports intestinal bacteria TOP10F '.Use Qiagen Tip 20 (Qiagen companies) from the intestinal bacteria transformant that obtains, to prepare plasmid DNA.Then, according to the incidental specification sheets of described test kit, use as primer-21M13 primer (Applied Biosystems Japanese firm) and M13Rev primer (Applied Biosystems Japanese firm), have the oligonucleotide and oligonucleotide of nucleotide sequence shown in the SEQ ID NO:56, stop cycle sequencing FS easy reaction test kit (Applied Biosystems Japanese firm) with dyestuff and carry out sequencing reaction with nucleotide sequence shown in the SEQ ID NO:64.With dna sequencing instrument 373A (Applied Biosystems Japanese firm) analytical reaction product.Based on the result, will have the plasmid called after pCR923 of nucleotide sequence shown in the SEQ ID NO:7 and will have the plasmid called after pCR923F of nucleotide sequence shown in the SEQ ID NO:10.

Next, each among usefulness restriction enzyme NdeI and HindIII digested plasmid pCR923 and the pCR923F.Digestion product is carried out agarose gel electrophoresis.Cutting comprises the gel area of about 1.2kbp DNA from the gel that carries out the pCR923 digestion product.Cutting comprises the gel area of about 1.4kbp DNA from the gel that carries out the pCR923F digestion product.By using the Qiagen PhastGel to extract test kit (Qiagen company) purify DNA from the gel of each recovery according to incidental specification sheets.Connect the DNA and the plasmid pKSN2 of the every kind of acquisition that digests with NdeI and HindIII and import e. coli jm109 with connecting test kit version 1 (Takara ShuzoCompany) according to the incidental specification sheets of described test kit.Prepare plasmid DNA from the intestinal bacteria transformant that obtains.Analyze its structure.Will be wherein between the NdeI site and HindIII site of about 1.2kbp DNA insertion pKSN2 of code book invention protein (A2), comprise the plasmid called after pKSN923 of nucleotide sequence shown in the SEQ ID NO:7.In addition, will be wherein between the NdeI site and HindIII site of about 1.4kbpDNA insertion pKSN2 of code book invention protein (A2), comprise the plasmid called after pKSN923F of nucleotide sequence shown in the SEQ ID NO:10.With each the importing e. coli jm109 among above-mentioned plasmid pKSN923 and the pKSN923F.With intestinal bacteria transformant difference called after JM109/pKSN923 and the JM109/pKSN923F that obtains.In addition, plasmid pKSN2 is imported e. coli jm109.The intestinal bacteria transformant called after JM109/pKSN2 that obtains.

(2) expression and the described recovery of protein of protein of the present invention (A2) in intestinal bacteria

With e. coli jm109/pKSN657, under each comfortable 37 ℃ of JM109/pKSN657F and the JM109/pKSN2 at the 10ml TB substratum that comprises 50 μ g/ml penbritins (1.2% (w/v) Tryptones, 2.4% (w/v) yeast extract, 0.4% (w/v) glycerine, the 17mM potassium primary phosphate, the 72mM dipotassium hydrogen phosphate) middle overnight incubation.The culture that one milliliter (1ml) obtained is transferred in the 100ml TB substratum that comprises 50 μ g/ml penbritins and cultivation under 26 ℃.When OD660 arrives approximately 0.5 the time, the 5-amino-laevulic acid is added to the final concentration of 500 μ M, continue to cultivate.After 30 (30) minutes,, further cultivated 17 hours the final concentration that IPTG adds to 1mM.

From each substratum, reclaim cell, wash and be suspended in the described damping fluid that 10ml comprises 1mM PMSF with 0.1Mtris-HCl damping fluid (pH7.5).Is 3 with the cell suspension that obtains in work output, carries out ultrasonoscope (Sonifer (Branson SonicPower Company)) under the condition of space factor 30% 6 times, each 3 minutes, so that obtain cell pyrolysis liquid.With cell pyrolysis liquid centrifugal (1,200xg, 5 minutes) back recovery supernatant liquor and centrifugal (150,000xg, 70 minutes) with reclaim the supernatant liquor fraction (below, the supernatant liquor fraction that will obtain from e. coli jm109/pKSN923 is called " intestinal bacteria pKSN923 extract ", the supernatant liquor fraction that will obtain from e. coli jm109/pKSN923F is called " intestinal bacteria pKSN923F extract ", and the supernatant liquor fraction that will obtain from e. coli jm109/pKSN2 is called " intestinal bacteria pKSN2 extract ").On 15%-25%SDS-PAGE, analyze a microlitre (1 μ l) above-mentioned supernatant liquor fraction and dye with CBB.As a result, all detect in intestinal bacteria pKSN923 extract and intestinal bacteria pKSN923F extract than intestinal bacteria pKSN2 extract intensive band significantly more, it is positioned at the electrophoresis position corresponding to the 47kDa molecular weight.Confirm that e. coli jm109/pKSN923 and e. coli jm109/pKSN923F express protein of the present invention (A2).

Prepare the reaction soln of 30 μ l and remain on 30 ℃ following 10 minutes.Reaction soln is made up of 0.1M potassium phosphate buffer (pH7.0), and it comprises 3ppm and uses ¹⁴The compound of C mark (II), 2mM β-NADPH (hereinafter referred to as " component A ") (Oriental Yeast Company), 0.2mg/ml derive from the ferredoxin (hereinafter referred to as " B component ") (Sigma Company) of spinach, the supernatant liquor fraction that 1U/ml ferredoxin reductase (hereinafter referred to as " component C ") (Sigma Company) and 18 μ l reclaim in embodiment 7 (2).In addition, prepare similarly and keep not to add be used for above-mentioned reaction soln composition, be selected from component A, the reaction soln of at least a component of B component and component C.Three microlitres (3 μ l) 2N HCl and 90 μ l ethyl acetate are added and be mixed in the every kind of reaction soln that keeps later.8, under the 000xg that the reaction soln that produces is centrifugal to reclaim 75 μ l ethyl acetate layers.After the drying under reduced pressure ethyl acetate layer, resistates is dissolved in the 6.0 μ l ethyl acetate.(5.0 μ l) puts silica gel tlc plate (TLC silica-gel plate 60F with its five microlitre ₂₅₄20cmx 20cm, 0.25mm is thick, Merck company) on.Use chloroform, the mixture of the 6:1:2 of acetate and ethyl acetate launched the TLC plate about 1 hour.Allow solvent evaporation then.The TLC plate is exposed to imaging plate (Fuji Film Company) to spend the night.Then, go up the analysis imaging plate at Image Analyzer BAS2000 (Fuji FilmCompany).Inspection is corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).The result is displayed in Table 8.

Table 8

The preparation of embodiment 8 protein of the present invention (A10)

(1) preparation of cell crude extract

With 250ml B substratum (1% (w/v) glucose in the freezing original seed adding 500ml band baffle plate flask of light gray streptomycete ATCC 11796,0.1% (w/v) extractum carnis, 0.2% (w/v) tryptose) and 30 ℃ of following rotational oscillation incubations 3 days to obtain pre-culture.40 milliliters (40ml) pre-culture added in the 400ml B substratum and under 30 ℃ in the 1L Erlenmeyer flask rotational oscillation incubation 24 hours.After stopping to cultivate, allow the culture sedimentation.Remove only ten milliliters of (220ml) supernatant liquors of mercurochrome.Ten milliliters of (220ml) fresh cultures of mercurochrome of similar preparation are added remaining 220ml substratum to amount to 440ml.To wherein adding the amount of compound (II) to 100ppm.Under 30 ℃ in the 1L Erlenmeyer flask rotational oscillation incubation cell 40 hours.Reclaim cell precipitation by the culture centrifugal (3,000g, 5 minutes) that 2.6L is produced.Wash the cell precipitation of generation so that 26g to be provided cell precipitation with 1L 0.1MPIPES-NaOH damping fluid (pH6.8).

With 1g cell precipitation 3ml these cell precipitations are suspended in the 0.1M PIPES-NaOH damping fluid (pH6.8), add 1mM PMSF, 5mM benzenyl amidine HCl, 1mMEDTA, 3 μ g/ml leupeptins, 3 μ g/ml pepstatin A and 1mM two sulphur tritols.By using French press (1000kg/cm ²) (Ohtake Seisakusho) break repeatedly suspension 2 times obtains cell pyrolysis liquid.After cell pyrolysis liquid is centrifugal (40,000xg, 30 minutes), reclaim supernatant liquor and 150, under the 000xg centrifugal 1 hour to reclaim supernatant liquor (hereinafter referred to as " cell crude extract ").

30 μ l reaction solns of preparation 0.1M potassium phosphate buffer (pH7.0), it comprises 3ppm and uses ¹⁴The compound of C mark (II), 2.4mM β-NADPH (hereinafter referred to as " component A ") (OrientalYeast Company), 0.5mg/ml derive from the ferredoxin (hereinafter referred to as " B component ") (Sigma Company) of spinach, the cell crude extract that 1U/ml ferredoxin reductase (hereinafter referred to as " component C ") (Sigma Company) and 18 μ l reclaim in embodiment 8 (1).With reaction soln remain on 30 ℃ following 1 hour.In addition, prepare similarly and keep not to add be used for above-mentioned reaction soln composition, be selected from component A, the reaction soln of at least a component of B component and component C.Three microlitres (3 μ l) 2N HCl and 90 μ l ethyl acetate are added and be stirred in the every kind of reaction soln that keeps later.8, under the 000xg that the reaction soln that produces is centrifugal to reclaim 75 μ l ethyl acetate layers.After the drying under reduced pressure ethyl acetate layer, resistates is dissolved in the 6.0 μ l ethyl acetate.(5.0 μ l) puts silica gel tlc plate (TLC silica-gel plate 60F with its five microlitre ₂₅₄20cm x20cm, 0.25 is thick, Merck company) on.Use chloroform, the mixture of the 6:1:2 of acetate and ethyl acetate launched the TLC plate about 1 hour.Allow solvent evaporation then.The TLC plate is exposed to imaging plate (FujiFilm Company) to spend the night.Then, go up the analysis imaging plate at Image Analyzer BAS2000 (Fuji FilmCompany).Inspection is corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).The result is displayed in Table 9.

Table 9

(3) fractional separation of cell crude extract

Ammonium sulfate is added in the saturated amount of cell crude extract to 45% that obtains among the embodiment 8 (1).Under ice-cooled condition, stir, by 12, reclaimed supernatant liquor under the 000xg in centrifugal 10 minutes.In the saturated amount of the supernatant liquor to 55% that ammonium sulfate add is obtained with after stirring under the ice-cooled condition, by 12, reclaimed precipitation under the 000xg in centrifugal 10 minutes.With the amount of two three propane damping fluid (pH7.0) dissolution precipitations of 20mM to 10ml.This solution is carried out PD10 post (AmershamPharmacia Company) and comprises proteinic fraction (hereinafter referred to as " 45-55% ammonium sulfate fraction ") with two three propane damping fluid (pH7.0) wash-outs of 20mM to reclaim 14ml.

(4) separation of protein of the present invention (A10)

The 45-55% ammonium sulfate fraction that will prepare in embodiment 8 (3) is expelled in MonoQHR 10/10 post (Amersham Pharmacia Company).Next, after the two three propane damping fluids (pH7.0) of the 20mM of 16ml flow to pillar, (gradient of NaCl is 0.00625M/ minute with the NaCl of linear gradient, the NaCl range of concentrations is 0M-0.5M, and flow velocity is 4ml/ minute) the two three propane damping fluids of the 20mM that flows together are recovered in the 15ml fraction of wash-out under the NaCl concentration of 0.28M-0.31M with classification.Further, the fraction that reclaims is carried out PD10 post (AmershamPharmacia Biotech Company) and comprise proteinic fraction with recovery with 20mM pair of three propane damping fluids (pH 7.0) wash-outs.

The fraction that reclaims is carried out PD10 post (Amersham Pharmacia Biotech Company), and (the 2mM potassium phosphate buffer that comprises 1.5mM NaCl, pH7.0) wash-out comprise proteinic fraction so that reclaim with buffer A.Next, fraction is injected among the Bio-Scale Ceramic hydroxylapatite I type pillar CHT10-I (BioRad Company).50 milliliters of (50ml) buffer A are flow to pillar.Subsequently, the buffer B of buffer A and linear gradient (the 100mM potassium phosphate buffer that comprises 0.03mMNaCl; Linear gradient increased to 50% buffer B since 100% buffer A during 40 minutes, flow velocity is 5ml/ minute) flowing together is recovered in the fraction of wash-out under the concentration of buffer B of 16%-31% with classification.Further, the fraction that reclaims is carried out PD10 post (Amersham Pharmacia Biotech Company) and comprised proteinic fraction with 0.05M potassium phosphate buffer (pH7.0) wash-out with recovery.Analysis package is contained in the protein in each fraction on 10%-20%SDS-PAGE.

Be similar to embodiment 8 (2), at component A, B component, component C and usefulness ¹⁴Under the existence of the composition of C mark (II), the fraction rather than the cell crude extract in embodiment 8 (2) described reaction solns that add and keep reclaiming.To keep later reaction soln TLC to analyze to check corresponding to usefulness ¹⁴The intensity of the spot of the compound of C mark (III).Observe the protein that in above-mentioned SDS-PAGE, moves to the 47kDa position and have the fluctuation that is parallel to corresponding to the strength fluctuation of the spot of compound (III) aspect the concentration of the fraction band that adds successively.From the SDS-PAGE gel, reclaim described protein and use tryptic digestion.At mass spectrograph (ThermoQuest Company, ion trap mass spectrometer LCQ, post: LC Packings Company PepMap C18 75 μ m x 150mm, solvent orange 2 A: 0.1% HOAc-H ₂O, solvent B:0.1%HOAc-methyl alcohol, gradient: begin in 30 minutes, to be increased to the linear gradient of the concentration of 100% solvent B, flow velocity: 0.2 μ l/ minute from mixture wash-out with 95% solvent orange 2 A and 5% solvent B) go up and analyze the digest that obtains.As a result, be provided at the aminoacid sequence of representing in each and any of SEQ ID NO:22-34.

The preparation of the chromosomal DNA of embodiment 9 light gray streptomycete ATCC 11796

At 50ml YEME substratum (0.3% (w/v) yeast extract, 0.5% (w/v) bactopeptone, 0.3% (w/v) wort, 1.0% (w/v) glucose, 34% (w/v) sucrose and 0.2% (v/v) 2.5MgCl ₂6H ₂O) in light gray streptomycete ATCC 11796 was descended the vibration incubations 1 day to 3 days at 30 ℃.Reclaim cell.With in the YEME substratum that comprises 1.4% (w/v) glycine and 60mM EDTA and the further vibration incubation 1 day of the cell suspension that obtains.From substratum, reclaim cell.With distilled water wash once after, with every 200mg cell 1ml it is resuspended in the damping fluid (100mM Tris-HCl (pH 8.0), 100mM EDTA, 10mM NaCl).The hen egg white lysozyme that adds every milliliter of two hectogamma (200 μ g/ml).Cell suspension was descended the vibration incubations 1 hour at 30 ℃.In addition, add 0.5%SDS and 1mg/ml Proteinase K.With cell suspension 55 ℃ of following incubations 3 hours.With phenol chloroform isoamyl alcohol extraction cell suspension 2 times to reclaim each water layer.Next, extract once to reclaim water layer with chloroform isoamyl alcohol.Obtain chromosomal DNA by the ethanol sedimentation water layer.

Embodiment 10 obtains the DNA of code book invention DNA (A10) and the expression in intestinal bacteria

(1) generation has the intestinal bacteria of the conversion of DNA of the present invention

By will in embodiment 9, carrying out PCR as template with by use Expand High Fidelity PCR system (Roche MolecularBiochemicals Company) by the chromosomal DNA from light gray streptomycete ATCC 11796 preparations.As primer, use oligonucleotide and have the pairing (hereinafter referred to as " primer is to 23 ") of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:80 or have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:79 and have the pairing (hereinafter referred to as " primer is to 24 ") of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:81 with nucleotide sequence shown in the SEQ ID NO:79.By adding 2 kinds every kind primer that amounts to 300nM, the above-mentioned chromosomal DNA of 50ng, 5.0 μ l dNTP mixtures (mixture of every kind of 2.0mM of 4 kinds of dNTP), 5.0 μ l, 10 x ExpandHF damping fluids (comprise MgCl ₂) and 0.75 μ l Expand HiFi enzyme mixture and distilled water, the PCR reaction soln amounts to 25 μ l.The reaction conditions of PCR is to remain on 97 ℃ after 2 minutes; Repeat 10 circulations, circulation comprise remain on 97 ℃ 15 seconds, then 65 ℃ 30 seconds, then 72 ℃ 2 minutes; Carry out 15 circulations then, circulation comprise remain on 97 ℃ 15 seconds, then 68 ℃ 30 seconds, then 72 ℃ 2 minutes (wherein each circulation increase remain on 72 ℃ following 20 seconds); Remain on then 72 ℃ 7 minutes.After maintenance, reaction soln is carried out 1% agarose gel electrophoresis.From the gel that uses primer that 23 reaction soln is carried out, reclaim the gel area that comprises about 1.2kbp DNA.From the gel that uses primer that 24 reaction soln is carried out, reclaim the gel area that comprises about 1.5kbp DNA.By using the Qiagen PhastGel to extract test kit (Qiagen company) purify DNA from the gel of each recovery according to incidental specification sheets.According to the incidental specification sheets of described carrier the DNA that obtains is connected to cloning vector pCR2.1-TOPO (Invitrogen company) and imports intestinal bacteria TOP10F '.Use Qiaprep Spin Miniprep test kit (Qiagen company) from the intestinal bacteria transformant that obtains, to prepare plasmid DNA.Next, according to the incidental specification sheets of described test kit, use as primer-21M13 primer (AppliedBiosystems Japanese firm), M13Rev primer (Applied Biosystems Japanese firm), have the oligonucleotide and oligonucleotide of nucleotide sequence shown in the SEQ ID NO:82, stop cycle sequencing FS easy reaction test kit (AppliedBiosystems Japanese firm) with dyestuff and carry out sequencing reaction with nucleotide sequence shown in the SEQ ID NO:83.Sequencing reaction uses the plasmid DNA that obtains as template.With dna sequencing instrument 373A (Applied Biosystems Japanese firm) analytical reaction product.Based on the result, will contain the plasmid called after pCR11796 and the plasmid called after pCR11796F that contains nucleotide sequence shown in the SEQ ID NO:85 of nucleotide sequence shown in the SEQ ID NO:84.There are two open reading-frame (ORF)s (ORF) in the described nucleotide sequence of in SEQ ID NO:85, representing.Therefore, comprise by 1221 Nucleotide (comprising terminator codon) and form and the nucleotide sequence (SEQ ID NO:84) of 406 amino-acid residues of encoding (aminoacid sequence of in SEQ ID NO:5, representing) and form and the nucleotide sequence of 69 amino-acid residues of encoding by 210 Nucleotide (comprising terminator codon).

Next, each among usefulness restriction enzyme NdeI and HindIII digestion pCR11796 and the pCR11796F.Digestion product is carried out agarose gel electrophoresis.Cutting comprises the gel area of about 1.2kbp DNA from the gel that carries out the pCR11796 digestion product.Cutting comprises the gel area of about 1.5kbp DNA from the gel that carries out the pCR11796F digestion product.By using the Qiagen PhastGel to extract test kit (Qiagen company) purify DNA from the gel of each recovery according to incidental specification sheets.Connect the DNA and the plasmid pKSN2 of the every kind of acquisition that digests with NdeI and HindIII and import e. coli jm109 with connecting test kit version 1 (Takara ShuzoCompany) according to the incidental specification sheets of described test kit.Prepare plasmid DNA from the intestinal bacteria transformant that obtains.Analyze its structure.Will be wherein between the NdeI site and HindIII site of about 1.2kbp DNA insertion pKSN2 of code book invention protein (A10), comprise the plasmid called after pKSN11796 of nucleotide sequence shown in the SEQ ID NO:84.In addition, will be wherein between the NdeI site and HindIII site of about 1.5kbp DNA insertion pKSN2 of code book invention protein (A10), comprise the plasmid called after pKSN11796F of nucleotide sequence shown in the SEQ ID NO:85.With each the importing e. coli jm109 among above-mentioned plasmid pKSN11796 and the pKSN11796F.With intestinal bacteria transformant difference called after JM109/pKSN11796 and the JM109/pKSN11796F that obtains.In addition, plasmid pKSN2 is imported e. coli jm109.The intestinal bacteria transformant called after JM109/pKSN2 that obtains.

(2) expression and the described recovery of protein of protein of the present invention (A10) in intestinal bacteria

With e. coli jm109/pKSN11796, under each comfortable 37 ℃ of JM109/pKSN11796F and the JM109/pKSN2 at the 10ml TB substratum that comprises 50 μ g/ml penbritins (1.2% (w/v) Tryptones, 2.4% (w/v) yeast extract, 0.4% (w/v) glycerine, the 17mM potassium primary phosphate, the 72mM dipotassium hydrogen phosphate) middle overnight incubation.The culture that one milliliter (1ml) obtained is transferred in the 100ml TB substratum that comprises 50 μ g/ml penbritins and cultivation under 26 ℃.When OD660 arrives approximately 0.5 the time, the 5-amino-laevulic acid is added to the final concentration of 500 μ M, continue to cultivate.After 30 (30) minutes,, further cultivated 17 hours the final concentration that IPTG adds to 1mM.

From each substratum, reclaim cell, wash and be suspended in the above-mentioned damping fluid that 10ml comprises 1mMPMSF with 0.1M tris-HCl damping fluid (pH7.5).The cell suspension that obtains in work output 3, is carried out ultrasonoscope (Sonifer (Branson SonicPower Company)) 6 times under the condition of space factor 30%, each 3 minutes, so that obtain cell pyrolysis liquid.With cell pyrolysis liquid centrifugal (1,200xg, 5 minutes) back recovery supernatant liquor and centrifugal (150,000xg, 70 minutes) with reclaim the supernatant liquor fraction (below, the supernatant liquor fraction that will obtain from e. coli jm109/pKSN11796 is called " intestinal bacteria pKSN11796 extract ", the supernatant liquor fraction that will obtain from e. coli jm109/pKSN11796F is called " intestinal bacteria pKSN11796F extract ", and the supernatant liquor fraction that will obtain from e. coli jm109/pKSN2 is called " intestinal bacteria pKSN2 extract ").On 15%-25%SDS-PAGE, analyze the above-mentioned supernatant liquor fraction of a microlitre (1 μ l) and use Coomassie blue (hereinafter referred to as " CBB ") dyeing.As a result, all detect in intestinal bacteria pKSN11796 extract and intestinal bacteria pKSN11796F extract than intestinal bacteria pKSN2 extract intensive band significantly more, it is positioned at the electrophoresis position corresponding to the 45kDa molecular weight.In intestinal bacteria pKSN11796F extract, identify than at intestinal bacteria pKSN11796 extract intensive band more.Show that e. coli jm109/pKSN11796F expresses protein of the present invention (A10) than e. coli jm109/pKSN11796 higher degree ground.

Prepare the reaction soln of 30 μ l and remain on 30 ℃ following 10 minutes.Reaction soln is made up of 0.1M potassium phosphate buffer (pH7.0), and it comprises 3ppm and uses ¹⁴The compound of C mark (II), 2mM β-NADPH (hereinafter referred to as " component A ") (Oriental Yeast Company), 2mg/ml derives from the ferredoxin (hereinafter referred to as " B component ") (Sigma Company) of spinach, the supernatant liquor fraction that 0.1U/ml ferredoxin reductase (hereinafter referred to as " component C ") (Sigma Company) and 18 μ l reclaim in embodiment 10 (2).In addition, prepare similarly and keep not to add be used for above-mentioned reaction soln composition, be selected from component A, the reaction soln of at least a component of B component and component C.Three microlitres (3 μ l) 2N HCl and 90 μ l ethyl acetate are added and be mixed in the every kind of reaction soln that keeps later.8, under the 000xg that the reaction soln that produces is centrifugal to reclaim 75 μ l ethyl acetate layers.After the drying under reduced pressure ethyl acetate layer, resistates is dissolved in the 6.0 μ l ethyl acetate.(5.0 μ l) puts silica gel tlc plate (TLC silica-gel plate 60F with its five microlitre ₂₅₄20cm x 20cm, 0.25mm is thick, Merck company) on.Use chloroform, the mixture of the 6:1:2 of acetate and ethyl acetate launched the TLC plate about 1 hour.Allow solvent evaporation then.The TLC plate is exposed to imaging plate (Fuji Film Company) to spend the night.Then, go up the analysis imaging plate at Image Analyzer BAS2000 (FujiFilm Company).Inspection is corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).The result is displayed in Table 10.

Table 10

Embodiment 11 obtains DNA of the present invention (A3)

(1) preparation of the chromosomal DNA of brick-red streptomycete ATCC 21469

At 50ml YEME substratum (0.3% (w/v) yeast extract, 0.5% (w/v) bactopeptone, 0.3% (w/v) wort, 1.0% (w/v) glucose, 34% (w/v) sucrose and 0.2% (v/v) 2.5MgCl ₂6H ₂O) in brick-red streptomycete ATCC 21469 was descended the vibration incubations 1 day to 3 days at 30 ℃.Reclaim cell.With in the YEME substratum that comprises 1.4% (w/v) glycine and 60mM EDTA and the further vibration incubation 1 day of the cell suspension that obtains.From substratum, reclaim cell.With distilled water wash once after, with every 200mg cell 1ml it is resuspended in the damping fluid (100mM Tris-HCl (pH 8.0), 100mM EDTA, 10mM NaCl).The hen egg white lysozyme that adds every milliliter of two hectogamma (200 μ g/ml).Cell suspension was vibrated 1 hour down at 30 ℃.In addition, add 0.5%SDS and 1mg/ml Proteinase K.With cell suspension 55 ℃ of following incubations 3 hours.With phenol chloroform isoamyl alcohol extraction cell suspension 2 times to reclaim each water layer.Next, extract once to reclaim water layer with chloroform isoamyl alcohol.Obtain chromosomal DNA by the ethanol sedimentation water layer.

(2) separation of DNA of the present invention (A3)

Carry out PCR by the chromosomal DNA that will in embodiment 11 (1), prepare as template.As primer, use oligonucleotide with nucleotide sequence shown in the SEQ ID NO:65 and pairing (hereinafter referred to as " primer is to 9 ") with oligonucleotide of nucleotide sequence shown in the SEQID NO:66.By adding the above-mentioned chromosomal DNA of 250ng, 2 kinds every kind primer that amounts to 200nM, 4 μ l dNTP mixtures (mixture of every kind of 2.5mM of 4 kinds of dNTP), 5 μ l, 10 x ExTaq damping fluids, 0.5 μ lExTaq polysaccharase (Takara Shuzo Company) and distilled water, the PCR reaction soln amounts to 50 μ l.The reaction conditions of PCR be remain on 97 ℃ 2 minutes; Repeat 30 circulations, circulation comprise remain on 97 ℃ 15 seconds, then 60 ℃ 30 seconds, then 72 ℃ 90 seconds; Remain on then 72 ℃ 4 minutes.After maintenance, reaction soln is carried out 0.8% agarose gel electrophoresis.Recovery comprises the gel area of the DNA of about 1.4kbp.By use the QIA PhastGel to extract test kit (Qiagen company) purify DNA from the gel that reclaims according to incidental specification sheets.According to the incidental specification sheets of described carrier the DNA that obtains is connected to TA cloning vector pCR2.1 (Invitrogen company) and imports intestinal bacteria TOP10F '.Use QIAprep Spin Miniprep test kit (Qiagen company) from the intestinal bacteria transformant that obtains, to prepare plasmid DNA.According to the incidental specification sheets of described test kit, the oligonucleotide that use has the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:67 and has nucleotide sequence shown in the SEQ ID NO:68 stops cycle sequencing FS easy reaction test kit (Applied Biosystems Japanese firm) with dyestuff and carries out sequencing reaction as primer.Sequencing reaction uses the plasmid that obtains as template.With dna sequencing instrument 373A (Applied Biosystems Japanese firm) analytical reaction product.As a result, be provided at the nucleotide sequence of representing among the SEQ ID NO:69.In described nucleotide sequence, there are two open reading-frame (ORF)s (ORF).Therefore, comprise by 1188 Nucleotide (comprising terminator codon) and form and the nucleotide sequence of 395 amino-acid residues of encoding and form and the nucleotide sequence (SEQID NO:17) of 64 amino-acid residues of encoding by 195 Nucleotide (comprising terminator codon).It is calculated that by the molecular weight of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:17 and to be 6666Da.

Embodiment 12 expression of protein of the present invention (A3) in intestinal bacteria

(1) contains the generation of the transformed into escherichia coli of DNA of the present invention (A3)

As above similarly carrying out PCR under the condition by the chromosomal DNA that will in embodiment 11 (1), prepare as template with by use ExTaq polysaccharase (Takara Shuzo Company).As primer, use oligonucleotide and have the pairing (hereinafter referred to as " primer is to 10 ") of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:71 or have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:70 and have the pairing (hereinafter referred to as " primer is to 11 ") of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:72 with nucleotide sequence shown in the SEQ ID NO:70.To be cloned among the TA cloning vector pCR2.1 the 1.5kbDNA of 11 amplifications the 1.2kb DNA and the use primer of 10 amplifications by using primer according to the method described above.Use QIAprep Spin Miniprep test kit (Qiagen company) from the intestinal bacteria transformant that obtains, to prepare plasmid DNA.According to the incidental specification sheets of described test kit, the oligonucleotide that use has the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:67 and has nucleotide sequence shown in the SEQ ID NO:68 stops cycle sequencing FS easy reaction test kit (Applied Biosystems Japanese firm) with dyestuff and carries out sequencing reaction as primer.Sequencing reaction uses the plasmid DNA that obtains as template.With dna sequencing instrument 373A (Applied Biosystems Japanese firm) analytical reaction product.As a result, confirm the plasmid of the dna clones of 10 amplifications to be contained nucleotide sequence shown in the SEQ ID NO:8 with primer.Confirmation contains nucleotide sequence shown in the SEQ ID NO:11 with primer to the plasmid of dna clones of 11 amplifications.There are two open reading-frame (ORF)s (ORF) in the described nucleotide sequence that in SEQID NO:11, shows.Therefore, comprise by 1188 Nucleotide (comprising terminator codon) and form and the nucleotide sequence (SEQ ID NO:8) of 395 amino-acid residues of encoding and form and the nucleotide sequence of 64 amino-acid residues of encoding by 195 Nucleotide (comprising terminator codon).The proteinic molecular weight of being made up of nucleotide sequence coded aminoacid sequence shown in the SEQ ID NO:8 it is calculated that and is 43752Da.As for the plasmid that obtains, will contain the plasmid called after pCR671 of nucleotide sequence shown in the SEQ ID NO:8 and will contain the plasmid called after pCR671F of nucleotide sequence shown in the SEQ ID NO:11.

Next, each among usefulness restriction enzyme NdeI and HindIII digestion pCR671 and the pCR671F.Digestion product is carried out agarose gel electrophoresis.Cutting comprises the gel area of about 1.2kbp DNA from the gel that carries out the pCR671 digestion product.Cutting comprises the gel area of about 1.5kbp DNA from the gel that carries out the pCR671F digestion product.By using the Qiagen PhastGel to extract test kit (Qiagen company) purify DNA from the gel of each recovery according to incidental specification sheets.Connect the DNA and the plasmid pKSN2 of the every kind of acquisition that digests with NdeI and HindIII and import e. coli jm109 with connecting test kit version 1 (Takara Shuzo Company) according to the incidental specification sheets of described test kit.Prepare plasmid DNA from the intestinal bacteria transformant that obtains.Analyze its structure.Will be wherein between the NdeI site and HindIII site of about 1200bp DNA insertion pKSN2 of code book invention protein (A3), comprise the plasmid called after pKSN671 of nucleotide sequence shown in the SEQ ID NO:8.In addition, will be wherein between the NdeI site and HindIII site of about 1400bp DNA insertion pKSN2 of code book invention protein (A3), comprise the plasmid called after pKSN671F of nucleotide sequence shown in the SEQ ID NO:11.With each the importing e. coli jm109 among above-mentioned plasmid pKSN671 and the pKSN671F.With intestinal bacteria transformant difference called after JM109/pKSN671 and the JM109/pKSN671F that obtains.In addition, plasmid pKSN2 is imported e. coli jm109.The intestinal bacteria transformant called after JM109/pKSN2 that obtains.

(2) expression and the described recovery of protein of protein of the present invention (A3) in intestinal bacteria

With e. coli jm109/pKSN671, under each comfortable 37 ℃ of JM109/pKSN671F and the JM109/pKSN2 at the 10ml TB substratum that comprises 50 μ g/ml penbritins (1.2% (w/v) Tryptones, 2.4% (w/v) yeast extract, 0.4% (w/v) glycerine, the 17mM potassium primary phosphate, the 72mM dipotassium hydrogen phosphate) middle overnight incubation.The culture that one milliliter (1ml) obtained is transferred in the 100ml TB substratum that comprises 50 μ g/ml penbritins and cultivation under 26 ℃.When OD660 arrives approximately 0.5 the time, the 5-amino-laevulic acid is added to the final concentration of 500 μ M, continue to cultivate.After 30 (30) minutes,, further cultivated 17 hours the final concentration that IPTG adds to 1mM.

From each substratum, reclaim cell, wash and be suspended in the described damping fluid that 10ml comprises 1mMPMSF with 0.1M tris-HCl damping fluid (pH7.5).The cell suspension that obtains in work output 3, is carried out ultrasonoscope (Sonifer (Branson SonicPower Company)) 6 times under the condition of space factor 30%, each 3 minutes, so that obtain cell pyrolysis liquid.With cell pyrolysis liquid centrifugal (1,200xg, 5 minutes) back recovery supernatant liquor and centrifugal (150,000xg, 70 minutes) with reclaim the supernatant liquor fraction (below, the supernatant liquor fraction that will obtain from e. coli jm109/pKSN671 is called " intestinal bacteria pKSN671 extract ", the supernatant liquor fraction that will obtain from e. coli jm109/pKSN671F is called " intestinal bacteria pKSN671F extract ", and the supernatant liquor fraction that will obtain from e. coli jm109/pKSN2 is called " intestinal bacteria pKSN2 extract ").

Prepare the reaction soln of 30 μ l and remain on 30 ℃ following 1 hour.Reaction soln is made up of 0.1M potassium phosphate buffer (pH7.0), and it comprises 3ppm and uses ¹⁴The compound of C mark (II), 2mM β-NADPH (hereinafter referred to as " component A ") (Oriental Yeast Company), 2mg/ml derives from the ferredoxin (hereinafter referred to as " B component ") (Sigma Company) of spinach, and the supernatant liquor level that 0.1U/ml ferredoxin reductase (hereinafter referred to as " component C ") (Sigma Company) and 18 μ l reclaim in embodiment 12 (2) is grouped into.In addition, prepare similarly and keep not to add be used for above-mentioned reaction soln composition, be selected from component A, the reaction soln of at least a component of B component and component C.Three microlitres (3 μ l) 2N HCl and 90 μ l ethyl acetate are added and be stirred in the every kind of reaction soln that keeps later.8, under the 000xg that the reaction soln that produces is centrifugal to reclaim 75 μ l ethyl acetate layers.After the drying under reduced pressure ethyl acetate layer, resistates is dissolved in the 6.0 μ l ethyl acetate.(5.0 μ l) puts silica gel tlc plate (TLC silica-gel plate 60F with its five microlitre ₂₅₄20cm x 20cm, 0.25mm is thick, Merck company) on.Use chloroform, the mixture of the 6:1:2 of acetate and ethyl acetate launched the TLC plate about 1 hour.Allow solvent evaporation then.The TLC plate is exposed to imaging plate (Fuji Film Company) to spend the night.Then, go up the analysis imaging plate at Image Analyzer BAS2000 (Fuji Film Company).Inspection is corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).The result is displayed in Table 11.

Table 11

Embodiment 13 obtains DNA of the present invention (A9)

(1) preparation of the chromosomal DNA of Streptomyces carbophilus SANK 62585

At 50ml YEME substratum (0.3% (w/v) yeast extract, 0.5% (w/v) bactopeptone, 0.3% (w/v) wort, 1.0% (w/v) glucose, 34% (w/v) sucrose and 0.2% (v/v) 2.5MgCl ₂6H ₂O) in Streptomyces carbophilus SANK 62585 (FERM BP-1145) was descended the vibration incubations 1 day at 30 ℃.Reclaim cell then.With in the YEME substratum that comprises 1.4% (w/v) glycine and 60mM EDTA and the further vibration incubation 1 day of the cell suspension that obtains.From substratum, reclaim cell.With distilled water wash once after, with every 200mg cell 1ml it is resuspended in the damping fluid (100mM Tris-HCl (pH 8.0), 100mM EDTA, 10mM NaCl).The hen egg white lysozyme that adds every milliliter of two hectogamma (200 μ g/ml).Cell suspension was vibrated 1 hour down at 30 ℃.In addition, add 0.5%SDS and 1mg/ml Proteinase K.With cell suspension 55 ℃ of following incubations 3 hours.With phenol chloroform isoamyl alcohol extraction cell suspension 2 times to reclaim each water layer.Next, extract once to reclaim water layer with chloroform isoamyl alcohol.Obtain chromosomal DNA by the ethanol sedimentation water layer.

(2) separation of DNA of the present invention (A9)

Carry out PCR by the chromosomal DNA that will in embodiment 13 (1), prepare as template.As primer, use oligonucleotide and have the pairing (hereinafter referred to as " primer is to 12 ") of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:75 or have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:76 and have the pairing (hereinafter referred to as " primer is to 13 ") of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:77 with nucleotide sequence shown in the SEQ ID NO:74.By adding 2 kinds every kind primer that amounts to 200nM, the above-mentioned chromosomal DNA of 250ng, 4 μ l dNTP mixtures (mixture of every kind of 2.5mM of 4 kinds of dNTP), 5 μ l 10x ExTaq damping fluids, 0.5 μ l ExTaq polysaccharase (Takara Shuzo Company) and distilled water, the PCR reaction soln amounts to 50 μ l.The reaction conditions of PCR be remain on 95 ℃ 2 minutes; Repeat 30 circulations, circulation comprise remain on 97 ℃ 15 seconds, then 60 ℃ 30 seconds, then 72 ℃ 90 seconds, remain on then 72 ℃ 4 minutes.After maintenance, reaction soln is carried out 0.8% agarose gel electrophoresis.From the gel that uses primer that 12 PCR reaction soln is carried out, reclaim the gel area of the DNA that comprises about 500bp.From the gel that uses primer that 13 PCR reaction soln is carried out, reclaim the gel area of the DNA that comprises about 800bp.By using the QIA PhastGel to extract test kit (Qiagen company) purify DNA from the gel of each recovery according to incidental specification sheets.According to the incidental specification sheets of described carrier the DNA that obtains is connected to TA cloning vector pCR2.1 (Invitrogen company) and imports intestinal bacteria TOP10F '.Use QIAprep Spin Miniprep test kit (Qiagen company) from the intestinal bacteria transformant that obtains, to prepare plasmid DNA.According to the incidental specification sheets of described test kit, the oligonucleotide that use has the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:67 and has nucleotide sequence shown in the SEQ ID NO:68 stops cycle sequencing FS easy reaction test kit (Applied Biosystems Japanese firm) with dyestuff and carries out sequencing reaction as primer.Sequencing reaction uses the plasmid DNA that obtains as template.With dna sequencing instrument 373A (Applied Biosystems Japanese firm) analytical reaction product.As a result, by using primer that the DNA that 12 PCR obtains is provided at the nucleotide sequence of representing among the Nucleotide 1-498 of nucleotide sequence shown in the SEQID NO:78.By using primer that the DNA that 13 PCR obtains is provided at the nucleotide sequence of representing among the Nucleotide 469-1233 of nucleotide sequence shown in the SEQ ID NO:78.The plasmid called after pCRSCA1 that will contain the nucleotide sequence of the Nucleotide 1-498 that in SEQ ID NO:78, represents.The plasmid called after pCRSCA2 that will contain the nucleotide sequence of the Nucleotide 469-1233 that in SEQ IDNO:78, represents.

Embodiment 14 expression of protein of the present invention (A9) in intestinal bacteria

(1) contains the generation of the transformed into escherichia coli of DNA of the present invention (A9)

The plasmid that use obtains in embodiment 13 (2) digests above-mentioned plasmid pCRSCA1 and digests pCRSCA2 with NdeI and NcoI with NdeI and NcoI.Digestion product is carried out agarose gel electrophoresis.Cutting comprises the gel area of about 500bp DNA from the gel that the pCRSCA2 digestion product carries out.Cutting comprises the gel area of about 800bp DNA from the gel that the pCRSCA2 digestion product carries out.By using the QIA PhastGel to extract test kit (Qiagen company) purify DNA from the gel of each recovery according to incidental specification sheets.Connect the DNA and the plasmid pKSN2 of two kinds of acquisitions that digest with NdeI and HindIII and import e. coli jm109 with connecting test kit version 1 (Takara Shuzo Company) according to the incidental specification sheets of described test kit.From the intestinal bacteria transformant that obtains, prepare plasmid DNA.Analyze its structure.Will be wherein the DNA of code book invention protein (A9) insert between the NdeI site and HindIII site of pKSN2, comprise the plasmid called after pKSNSCA of nucleotide sequence shown in the SEQ ID NO:78.

(2) expression and the described recovery of protein of protein of the present invention (A9) in intestinal bacteria

With e. coli jm109/pKSNSCA under 37 ℃ at the 10mlTB substratum that comprises 50 μ g/ml penbritins (1.2% (w/v) Tryptones, 2.4% (w/v) yeast extract, 0.4% (w/v) glycerine, 17mM potassium primary phosphate, 72mM dipotassium hydrogen phosphate) middle overnight incubation.The culture that obtains is transferred in the 100ml TB substratum that comprises 50 μ g/ml penbritins and at 26 ℃ to descend to cultivate so that OD660 is 0.2.When OD660 arrives approximately 2.0 the time, the 5-amino-laevulic acid is added to the final concentration of 500 μ M, continue to cultivate.After 30 (30) minutes,, further cultivated 5 hours the final concentration that IPTG adds to 200 μ M.

From each substratum, reclaim cell, wash and be suspended in the described damping fluid that 10ml comprises 1mM PMSF with 0.1M tris-HCl damping fluid (pH7.5).The cell suspension that obtains in work output 3, is carried out ultrasonoscope (Sonifer (Branson SonicPower Company)) 6 times under the condition of space factor 30%, each 3 minutes, so that obtain cell pyrolysis liquid.With cell pyrolysis liquid centrifugal (1,200xg, 5 minutes) back reclaim supernatant liquor and centrifugal (150,000xg, 70 minutes) to reclaim supernatant liquor fraction (below, the supernatant liquor fraction that will obtain is called " intestinal bacteria pKSNSCA extract ") from e. coli jm109/pKSNSCA.

Prepare the reaction soln of 30 μ l and remain on 30 ℃ following 10 minutes.Reaction soln is made up of 0.1M potassium phosphate buffer (pH7.0), and it comprises 3ppm and uses ¹⁴The compound of C mark (II), 2mM β-NADPH (hereinafter referred to as " component A ") (Oriental Yeast Company), 2mg/ml derives from the ferredoxin (hereinafter referred to as " B component ") (Sigma Company) of spinach, the supernatant liquor fraction that 0.1U/ml ferredoxin reductase (hereinafter referred to as " component C ") (Sigma Company) and 18 μ l reclaim in embodiment 14 (2).In addition, prepare similarly and keep not to add be used for above-mentioned reaction soln composition, be selected from component A, the reaction soln of at least a component of B component and component C.Three microlitres (3 μ l) 2N HCl and 90 μ l ethyl acetate are added and be stirred in the every kind of reaction soln that keeps later.8, under the 000xg that the reaction soln that produces is centrifugal to reclaim 75 μ l ethyl acetate layers.After the drying under reduced pressure ethyl acetate layer, resistates is dissolved in the 6.0 μ l ethyl acetate.(5.0 μ l) puts silica gel tlc plate (TLC silica-gel plate 60F with its five microlitre ₂₅₄20cm x 20cm, 0.25mm is thick, Merck company) on.Use chloroform, 6: 1: 2 mixture of acetate and ethyl acetate launched the TLC plate about 1 hour.Allow solvent evaporation then.The TLC plate is exposed to imaging plate (Fuji Film Company) to spend the night.Then, go up the analysis imaging plate at Image Analyzer BAS2000 (FujiFilm Company).Inspection is corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).The result is displayed in Table 12.

Table 12

The separation of embodiment 15 soybean RuBPC genes

Behind sowing soybean (cv.Jack), cultivated soybean 30 days and collected leaf down at 27 ℃.Also grind with liquid nitrogen freezing 2/10ths grams (0.2g) to the leaf that 0.3g collects with mortar and pestle.Subsequently, use RNA to extract solvent ISOGEN (Nippon Gene Company) according to incidental handbook and from the ground product, extract total RNA.Further, be used for the synthetic cDNA of Superscript article one chain synthesis system (Invitrogen Company) of RT-PCR by carry out program according to incidental handbook.Particularly, by the widow (dT) who provides by test kit is provided _12-18Primer as primer and total soybean RNA as template with by to the synthetic article one chain cDNA of the reversed transcriptive enzyme that provides by test kit wherein is provided.Next, by pcr amplification coding back 12 amino acid whose soybean of maturation protein (cv.Jack) ribulose-1,5-bisphosphate is arranged, 5-bisphosphate carboxylase is (following with ribulose-1,5-bisphosphate, 5-bisphosphate carboxylase is called " RuBPC ") DNA of small subunit chloroplast transit peptides (below, soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides is sometimes referred to as " rSt "; The coding back has the DNA of 12 amino acid whose soybean of maturation protein (cv.Jack) RuBPC small subunit chloroplast transit peptides to be called " rSt12 DNA of the present invention ").The cDNA that PCR use to obtain is as template and have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:86 and have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:87 as primer.PCR uses LA Taq polysaccharase (Takara Shuzo Company).By keep 94 ℃ 3 minutes 1 time; Carry out 30 circulations, circulation comprise keep 98 ℃ 25 seconds and then 68 ℃ 1 minute; With keep 72 ℃ to carry out PCR 10 minutes 1 time.Insert acquisition plasmid pCRrSt12 (Fig. 5) in plasmid pCR2.1 (Invitrogen Company) the PCR product cloning site by DNA with amplification.Next, plasmid is imported in the competent cell of e. coli jm109 bacterial strain and select anti-penbritin bacterial strain.In addition, by using dyestuff termination cycle sequencing FS easy reaction test kit (PE Applied Biosystems Company) and dna sequencing instrument 373S (PE Applied Biosystems Company) to measure the nucleotide sequence that is included in the plasmid in the selected anti-penbritin bacterial strain.As a result, be provided at the nucleotide sequence of representing among the SEQ ID NO:88.Confirm that plasmid pCRrSt12 comprises rSt12DNA of the present invention.

Embodiment 16 is used for the directly structure of the chloroplast expression plasmid that comprises DNA of the present invention (A1) of importing

(1) separation of DNA of the present invention (A1)

The DNA that comprises nucleotide sequence shown in the SEQ ID NO:6 by pcr amplification.Genomic dna by using actinomyces abortion within the first month of pregnancy look streptomycete IFO 12898 is as template with by using oligonucleotide of being made up of nucleotide sequence shown in the SEQ ID NO:93 and the oligonucleotide of being made up of nucleotide sequence shown in the SEQ ID NO:94 to carry out PCR as primer.In addition, the DNA that comprises nucleotide sequence shown in the SEQ ID NO:9 by pcr amplification.Oligonucleotide sequence carries out PCR as primer shown in the oligonucleotide be made up of nucleotide sequence shown in the SEQ ID NO:93 and the SEQ ID NO:95 by using.Described PCR uses Expand High Fidelity PCR System (BoehringerCompany).Keeping 97 ℃ after 2 minutes 1 time; Carry out 10 circulations, circulation comprise keep 97 ℃ 15 seconds, then 60 ℃ 30 seconds and then 72 ℃ 1 minute; Carry out 15 circulations then, circulation comprise keep 97 ℃ 15 seconds, then 60 ℃ of 30 seconds and then 72 ℃ 1 minute (wherein each circulates in 72 ℃ maintenance increases by 20 seconds); Keep 72 ℃ to carry out PCR in 7 minutes then.The PCR product cloning zone of inserting pCR2.1 (Invitrogen Company) by the DNA with amplification produces plasmid pCR657ET (Fig. 6) and pCR657FET (Fig. 7).In addition, except the oligonucleotide that uses the oligonucleotide formed by nucleotide sequence shown in the SEQ ID NO:96 and form by nucleotide sequence shown in the SEQ IDNO:94, to be similar to the program acquisition plasmid pCR657Bs (Fig. 8) of aforesaid method.Further again, except the oligonucleotide that uses the oligonucleotide formed by nucleotide sequence shown in the SEQ ID NO:96 and form by nucleotide sequence shown in the SEQ ID NO:97, to be similar to the program acquisition plasmid pCR657FBs (Fig. 9) of aforesaid method.Next, plasmid is imported bacillus coli DH 5 alpha competent cell (Takara Shuzo Company) and select the cell of anti-penbritin.In addition, by using BigDye to stop the nucleotide sequence that cycle sequencing easy reaction test kit 2.0 editions (PE Applied Biosystems Company) and dna sequencing instrument 3100 (PE Applied Biosystems Company) mensuration are included in the plasmid in the anti-penbritin bacterial strain.As a result, confirm that plasmid pCR657ET and pCR657Bs contain nucleotide sequence shown in the SEQ ID NO:6.Confirm that plasmid pCR657FET and pCR657FBs contain nucleotide sequence shown in the SEQ ID NO:9.

(2) be used for directly structure-partly (1) of the chloroplast expression plasmid that comprises DNA of the present invention (A1) of importing

The plasmid that structure comprises chimeric DNA is as importing the plasmid of plant with particle bombardment with DNA of the present invention (A1), and DNA of the present invention (A1) is right after the variation that nucleotide sequence at coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides (below be sometimes referred to as " sequence of coding chloroplast transit peptides ") does not have the codon frame afterwards in the described chimeric DNA.

At first, with restriction enzyme HindIII and KpnI digestion pCRrSt12.Separate the DNA that comprises rSt12DNA of the present invention.In addition, by from plasmid vector pUC19 (Takara Shuzo Company), removing the DNA that about 40bp DNA obtains about 2640bp with restriction enzyme HindIII and KpnI digestion.Next, with 5 ' the terminal dephosphorylation of calf intestinal alkaline phosphatase (Takara Shuzo Company) with DNA.To wherein inserting DNA that obtain, that comprise rSt12DNA of the present invention, obtain pUCrSt12 (Figure 10) from pCRrSt12.Next, by separating the DNA that comprises DNA of the present invention (A1) with among the pCR657FET each with restriction enzyme EcoT22I and SacI digested plasmid pCR657ET.Among the DNA that obtains each is inserted between the EcoT22I restriction site of pUCrSt12 and the SacI restriction site obtaining to comprise plasmid pUCrSt657 (Figure 11) and the pUCrSt657F (Figure 12) of chimeric DNA, and DNA of the present invention (A1) is right after after the nucleotide sequence of soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides of encoding and does not have a variation of codon frame in the described chimeric DNA.

Digest pBICR16G6PT (in the patent 2000-166577 of Japanese unexamined, describing) to separate the DNA of about 3kb with limiting enzyme EcoRI.(below, the promotor that is included among the described DNA of top Japanese unexamined patent is called " CR16G6 promotor ".In addition, the terminator that is included among the described DNA of top Japanese unexamined patent is called " CR16G6 terminator ".) after with limiting enzyme EcoRI digested plasmid carrier pUC19 (Takara Shuzo Compnay), use 5 ' the terminal dephosphorylation of calf intestinal alkaline phosphatase (Takara Shuzo Company) with described DNA.To wherein inserting available from the 3kb DNA of pBICR16G6PT to obtain plasmid pUCCR16G6-p/t (Figure 13).Digest pUCCR16G6-p/t to separate the DNA that comprises the CR16G6 promotor with restriction enzyme HindIII and ScaI.In addition, by with restriction enzyme HindIII and EcoRI digested plasmid carrier pUC19 (Takara Shuzo Company), remove the DNA of 51bp and the surplus DNA that acquisition is made up of 2635bp.Next, with 5 ' the terminal dephosphorylation of calf intestinal alkaline phosphatase (Takara ShuzoCompany) with described DNA.Comprise available from the above-mentioned DNA of the CR16G6 promotor of pUCCR16G6-p/t and NotI-EcoRI joint (Figure 14) obtaining pUCCR12G6-p/t Δ (Figure 15) to wherein inserting, described NotI-EcoRI joint is annealed available from the oligonucleotide that will be made up of nucleotide sequence shown in the SEQ IDNo:89 with by the oligonucleotide that nucleotide sequence shown in the SEQ ID No:90 is formed.Digest the pUCCR12G6-p/t Δ to separate the DNA of the partial nucleotide sequence that contains the CR16t terminator with restriction enzyme NdeI and EcoRI.In addition, use restriction enzyme HindIII and EcoRI digested plasmid carrier pUC19 (Takara Shuzo Company) to obtain the DNA of 2635bp.With 5 ' the terminal dephosphorylation of calf intestinal alkaline phosphatase (Takara Shuzo Company) with described DNA.Have available from the above-mentioned DNA of the partial nucleotide sequence of the CR16t terminator of pUCCR12G6-p/t Δ and HindIII-NotI joint (Figure 16) obtaining pNdG6-Δ T (Figure 17) to wherein inserting, described HindIII-NotI joint is annealed available from the oligonucleotide that will be made up of nucleotide sequence shown in the SEQ IDNo:91 with by the oligonucleotide that nucleotide sequence shown in the SEQ ID No:92 is formed.

Next, by using each among restriction enzyme BamHI and SacI digested plasmid pUCrSt657 and the pUCr657F, separate the DNA comprise chimeric DNA, DNA of the present invention (A1) is right after after the nucleotide sequence of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and does not have a variation of codon frame in chimeric DNA.DNA is inserted between the BglII restriction enzyme sites of plasmid pNdG6-Δ T and the SacI restriction enzyme sites to obtain each among plasmid pSUM-NdG6-rSt-657 (Figure 18) and the plasmid pSUM-NdG6-rSt-657F (Figure 19).

(3) be used for directly structure-partly (2) of the chloroplast expression plasmid with DNA of the present invention (A1) of importing

The plasmid that structure comprises chimeric DNA is as importing the plasmid of plant with particle bombardment with DNA of the present invention (A1), and DNA of the present invention (A1) is right after after 12 amino acid whose nucleotide sequences of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and the mature protein thereafter of encoding and does not have a variation of codon frame in the described chimeric DNA.At first, after with restriction enzyme BspHI digested plasmid carrier pKF19 (Takara Shuzo Company), Nucleotide is added double-stranded breach with the terminal flush endization of DNA by using KOD archaeal dna polymerase (Toyobo Corporation).By the DNA recirculation that obtains being obtained plasmid pKF19 Δ Bs with the T4DNA ligase enzyme.The pCRrSt12 of acquisition in embodiment 1 with restriction enzyme HindIII and KpnI digestion.Separate the DNA that comprises rSt12DNA of the present invention.With restriction enzyme HindIII and KpnI digested plasmid pKF19 Δ Bs to obtain the DNA of about 2160bp.With 5 ' the terminal dephosphorylation of calf intestinal alkaline phosphatase (Takara Shuzo Cormpany) with described DNA.To wherein inserting the DNA comprise available from the rSt12DNA of the present invention of pCRrSt12 to obtain pKFrSt12 (Figure 20).Next, with among the plasmid pCR657Bs of restriction enzyme BspHI and SacI digestion acquisition among embodiment 16 (1) and the PCR657FBs each to separate the DNA that comprises DNA of the present invention (A1).Among these DNA each is inserted between the BspHI restriction site of plasmid pKFrSt12 and the SacI restriction site to obtain plasmid pKFrSt12-657 (Figure 21) and plasmid pKFrSt12-657F (Figure 22), it comprises chimeric DNA, and DNA of the present invention (A1) is right after after 12 amino acid whose nucleotide sequences of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and the mature protein thereafter of encoding and does not have a variation of codon frame in described chimeric DNA.

Next, with among BamHI and SacI digested plasmid pKFrSt12-657 and the pKFrSt12-657F each to obtain to comprise the DNA of DNA of the present invention (A1).Among these DNA each is inserted between the BglII restriction site and SacI restriction site of the plasmid pNdG6-Δ T that obtain among the embodiment 16 (2), to obtain plasmid pSUM-NdG6-rSt12-657 (Figure 23) and pSUM-NdG6-rSt12-657F (Figure 24), wherein chimeric DNA is connected the downstream of promotor CR16G6, and DNA of the present invention (A1) is right after after 12 amino acid whose nucleotide sequences of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and the mature protein thereafter of encoding and does not have a variation of codon frame in described chimeric DNA.

Embodiment 17 imports soybean with DNA of the present invention (A1)

(1) preparation of vegetation somatic embryo

After soybean (Cultivar: Fayette and Jack) pod being dipped in 1% chlorine bleach liquor sterilization, take out immature seed.Kind of skin is peeled off the immature embryo that has the 2-5mm diameter to remove from seed.The plumular axis of the immature embryo that obtains with scalper cutting is with the preparation unmature subleaf.Unmature subleaf is divided into 2 cotyledon parts.Each cotyledon part is put in respectively in the somatic embryo development substratum.The somatic embryo development substratum is a solid medium, wherein 0.2% (w/v) being taken off the adding of acetyl gellan gum is set at pH7.0 and wherein adds 180 μ M 2, the Murashige-Skoog substratum of 4-D and 30g/L sucrose is (at Murashige T. and Skoog F., Physiol.Plant (1962) 15, the 473rd page; Hereinafter referred to as " MS substratum ").After placing 1 month, the globular embryo that forms is transplanted in the somatic embryo growth medium.The somatic embryo growth medium is a solid medium, wherein 0.2% (w/v) is taken off the adding of acetyl gellan gum and is set at pH5.8 and wherein adds 90 μ M2, the MS substratum of 4-D and 30g/L sucrose.With the interval in 2-3 week globular embryo be transplanted in fresh somatic embryo growth medium 5-8 time thereafter.The culture condition separately that uses above-mentioned somatic embryo development substratum and somatic embryo growth medium is that illumination in 23 hours and 1 hour are dark and whole day 23-25 ℃.

(2) gene is imported the vegetation somatic embryo

Embodiment 17 (1) globular embryos that obtain are being transplanted in the fresh somatic embryo growth medium and after cultivating 2-3 days, globular embryo are being used for quiding gene.With plasmid pSUM-NdG6-rSt657, pSUM-NdG6-rSt657F, pSUM-NdG6-rSt12657 and pSUM-NdG6-rSt12657F are coated on the gold grain of diameter 1.0 μ m and import with the gene that uses particle bombardment.Amount for the plasmid of 1mg gold grain is 1.66 μ g.Behind quiding gene, embryo was further cultivated 2-3 days.Respectively do for oneself illumination in 23 hours and 1 hour of culture condition is dark and whole day 23-25 ℃.

(3) with Totomycin selective body somatic embryo

To be transplanted to somatic embryo at the later globular embryo of quiding gene that embodiment 17 (2) obtains and select substratum.It is solid medium that somatic embryo is selected substratum, wherein 0.2% (w/v) is taken off the adding of acetyl gellan gum and 15mg/L Totomycin and is set at pH5.8 and has wherein added 90 μ M2, the MS substratum of 4-D and 30g/L sucrose.After this interval with 2-3 week migrates to fresh somatocyte selection substratum 5-8 time with the globular embryo of surviving.At that time, it is solid medium that somatic embryo is selected substratum, wherein 0.2% (w/v) is taken off the adding of acetyl gellan gum and 30mg/L Totomycin and is set at pH5.8 and wherein adds 90 μ M 2, the MS substratum of 4-D and 30g/L sucrose.Use above-mentioned somatic embryo to select respectively do for oneself illumination in 23 hours and 1 hour of the culture condition of substratum dark and whole day 23-25 ℃.

(4) with compound (II) selective body somatic embryo

To be transplanted to somatic embryo at the later globular embryo of quiding gene that embodiment 17 (2) obtains and select substratum.It is solid medium that somatic embryo is selected substratum, wherein 0.2% (w/v) is taken off the adding of acetyl gellan gum and 0.1mg/L compound (II) and is set at pH5.8 and has wherein added 90 μ M2, the MS substratum of 4-D and 30g/L sucrose.After this interval with 2-3 week migrates to fresh somatocyte selection substratum 5-8 time with the globular embryo of surviving.At that time, it is solid medium that somatic embryo is selected substratum, wherein 0.2% (w/v) is taken off the adding of acetyl gellan gum and 0.3-1mg/L compound (II) and is set at pH5.8 and has wherein added 90 μ M2, the MS substratum of 4-D and 30g/L sucrose.Use above-mentioned somatic embryo to select respectively do for oneself illumination in 23 hours and 1 hour of the culture condition of substratum dark and whole day 23-25 ℃.

(5) from the plant regeneration of somatic embryo

The globular embryo that to select in embodiment 17 (3) or 17 (4) is transplanted to and is grown in the substratum and dark and whole day 23-25 ℃ of 4 weeks of cultivation down illumination in 23 hours and 1 hour.Growing substratum is solid medium, wherein 0.8% (w/v) agar (Wako Pure Chemical Industries, Ltd. is used for plant tissue culture) is added the MS substratum that is set at pH5.8 and has wherein added 60g/L maltose.Thereafter 6-8 week obtains the embryo of white to yellow cotyledon type.With the embryonic implantation of these cotyledon types to the substratum that germinates and cultivated for 2 weeks.The germination substratum is a solid medium, wherein 0.2% (w/v) is taken off the acetyl gellan gum and adds the MS substratum that is set at pH5.8 and has wherein added 30g/L sucrose.As a result, the soybean that can obtain to have grown leaf and have root.

(6) aftergrowth complying with and cultivating

The soybean that will obtain in embodiment 17 (5) is transplanted to plough in soil and dark illumination in 23 hours and 1 hour in whole day 23-25 ℃ the brooder house and complies with.Two (2) Zhou Yihou, the plant that will take root is transferred in the basin that diameter is 9cm and at room temperature cultivates.Culture condition at room temperature is 23 ℃-25 ℃ of whole day natural lights.Two to four (2-4) collected soybean seeds after individual month.

(7) resistance of herbicidal compound (II) is assessed

Collect the leaf of aftergrowth and be divided into 2 along the main lobe arteries and veins.Compound (II) is dispersed on the whole surface of one of blade.Another blade stays and is untreated.These blades are placed on the MS substratum that comprises 0.8% agar and at room temperature were positioned in the light 7 days.Then, in the 5ml80% aqueous acetone solution, grind each blade to extract chlorophyll with pestle and mortar.With 80% aqueous acetone solution, 10 times of extracting solutions of dilution with according to Mackenney, G., J.Biol.Chem. (1941) 140; The 315th page of described method is at 750nm, and 663nm and 645nm measure absorbancy down to calculate total chlorophyll content.The percentile of total chlorophyll content of the blade by display process and total chlorophyll content of the blade that is untreated can comparative assessment to the degree of the resistance of compound (II).

Then, soil being clogged to diameter is that the 10cm and the degree of depth are in the plastic tub of 10cm.Sow the seed of above-mentioned plant and in the greenhouse, cultivate.By mixing 5 parts of compounds (II), 6 parts of Sorpols (sorpol) 3005X (Toho chemical) and 89 parts of dimethylbenzene prepare emulsion.Be dispersed in equably on all faces from the leaf of the top plant of in above-mentioned basin, cultivating with the emulsion of the dilution proportion specified quantitative of 1 hectare of 1000L and with spray gun with the water that comprises 0.1% (v/v) tackiness agent.Culturing plants is after 16 days in the greenhouse, and research is to the damage of plant, and assessment is to the resistance of compound (II).

Embodiment 18 is used for the structure of the chloroplast expression plasmid with DNA of the present invention (A1) of Agrobacterium importing

Structure imports DNA of the present invention (A1) with agrobacterium co-cultivation the plasmid of plant.At first, after with restriction enzyme NotI digestion binary plasmid (binary plasmid) carrier pBI121 (ClontechCompany), Nucleotide is added double-stranded breach with the terminal flat endization of DNA by using dna polymerase i (Takara Shuzo Corporation).The T4DNA ligase enzyme is used for recirculation.After the plasmid that obtains with limiting enzyme EcoRI digestion, Nucleotide is added double-stranded breach with the terminal flat endization of DNA by using dna polymerase i (Takara Shuzo Corporation).The T4DNA ligase enzyme is used for recirculation to obtain plasmid pBI121 Δ NotIEcoRI.After with the HindIII digested plasmid, with 5 ' the DNA end dephosphorylation of calf intestinal alkaline phosphatase (Takara Shuzo Company) with the DNA of acquisition.The HindIII-NotI-EcoRI joint (Figure 25) that obtains to the oligonucleotide annealing that wherein is inserted through the oligonucleotide that to form by nucleotide sequence shown in the SEQ ID NO:98 and forms by nucleotide sequence shown in the SEQ ID NO:99.Obtain binary plasmid carrier pBI121S (Figure 26) by recirculation.Described plasmid has the HindIII-NotI-EcoRI joint with HindIII restriction site wherein, the structure that the direction that NotI restriction site and EcoRI restriction site are arranged in order from the position near beta-Glucuronidase is inserted.

Next, with among restriction enzyme HindIII and EcoRI digested plasmid pSUM-NdG6-rSt-657 and the pSUM-NdG6-rSt-657F each, so that each obtains chimeric DNA from it, DNA wherein of the present invention (A1) is right after after the nucleotide sequence of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and does not have a variation of codon frame.These DNA are inserted between the HindIII restriction site of above-mentioned binary plasmid carrier pBI121S and the EcoRI restriction site to obtain plasmid pBI-NdG6-rSt-657 (Figure 27) and pBI-NdG6-rSt-657F (Figure 28).In addition, with each usefulness restriction enzyme HindIII among above-mentioned plasmid pSUM-NdG6-rSt12-657 and the pSUM-NdG6-rSt12-657F and EcoRI digestion, obtaining chimeric DNA from each, DNA wherein of the present invention (A1) is right after after 12 amino acid whose nucleotide sequences of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and the mature protein thereafter of encoding and does not have a variation of codon frame.These DNA are inserted between the HindIII restriction site of above-mentioned binary plasmid carrier pBI121S and the EcoRI restriction site to obtain plasmid pBI-NdG6-rSt12-657 (Figure 29) and pBI-NdG6-rSt12-657F (Figure 30).

Embodiment 19 imports tobacco with DNA of the present invention (A1)

The plasmid pBI-NdG6-rSt-657 that use obtains in embodiment 18, plasmid pBI-NdG6-rSt-657F, plasmid pBI-NdG6-rSt12-657 and plasmid pBI-NdG6-rSt12-657F import tobacco with agrobacterium co-cultivation with DNA of the present invention (A1).

At first, respectively with plasmid pBI-NdG6-rSt-657, pBI-NdG6-rSt-657F, pBI-NdG6-rSt12-657 and pBI-NdG6-rSt12-657F import agrobacterium tumefaciens (Agrobacterium tumefaciens) LBA4404 (Clontech Company).Containing the 300mg/L Streptomycin sulphate by the transformant that will produce, LB nutrient agar (0.5% yeast extract of 100mg/L Rifampin and 25mg/L kantlex, 1.0% bacto-tryptone, cultivate 0.5%NaCl) and by selecting resistance clone, separate and contain pBI-NdG6-rSt-657, pBI-NdG6-rSt-657F, the agrobacterium strains of the conversion of pBI-NdG6-rSt12-657 or pBI-NdG6-rSt12-657F.

Then, according to the described method of plant gene operational manual (Hirofumi UCHIMIYA, Kodan-shaScientific, 1992), gene is imported tobacco.To contain under each comfortable 28 ℃ of the Agrobacterium bacterial strains of above-mentioned plasmid and contain the 300mg/L Streptomycin sulphate, overnight incubation in the LB substratum of 100mg/L Rifampin and 25mg/L kantlex, the blade with the tobacco of sterile culture immerses in the liquid nutrient medium then.Comprising MS nutrient agar (the MS inorganic salt of 0.1mg/L naphthylacetic acid and 1.0mg/L benzyladenine in plantation blade and the illumination at room temperature, the MS VITAMIN, 3% sucrose and 0.8% agar, at Murashige T. and Skoog F., Physiol.Plant. describe in the (1962) 15, the 473rd page) in cultivated 2 days.Then, with the sterilized water washing blade and containing the 0.1mg/L naphthylacetic acid, cultivated 7 days on the MS nutrient agar of 1.0mg/L benzyladenine and 500mg/L cefotaxime.Next, transplant blade and containing the 0.1mg/L naphthylacetic acid, the 1.0mg/L benzyladenine is cultivated in the MS nutrient agar of 500mg/L cefotaxime and 100mg/L kantlex.Cultivate and carried out 4 months continuously, with the interval in 4 weeks blade is transferred to the fresh culture of same composition simultaneously.At that time, transplant from the unfixed bud of leaf development and take root in the MS nutrient agar that comprises 300mg/L cefotaxime and 50mg/L kantlex to obtain regenerate.Regenerate is transplanted and cultivated in comprising the MS nutrient agar of 50mg/L kantlex, to obtain wherein to have imported pBI-NdG6-rSt-657 respectively, pBI-NdG6-rSt-657F, the transgene tobacco in the T-DNA zone of pBI-NdG6-rSt12-657 or pBI-NdG6-rSt12-657F.

In addition, the plasmid pBI121S that will obtain in embodiment 18 with agrobacterium co-cultivation imports tobacco.Except using plasmid pBI121S rather than pBI-NdG6-rSt-657, pBI-NdG6-rSt-657F beyond pBI-NdG6-rSt12-657 and the pBI-NdG6-rSt12-657F, separates the Agrobacterium bacterial strain of the conversion that contains pBI121S above being similar to.Next, utilize the Agrobacterium of described conversion, be similar to the transgene tobacco that above-mentioned acquisition has imported the T-DNA zone of plasmid pBI121S.

Get three (3) sheet leaves from transgene tobacco.Every leaf is divided into 4, and wherein every 5-7mm is wide.Plant every leaf on the MS nutrient agar that contains 0.1mg/L compound (II) and cultivation at room temperature in illumination.At the 7th day that cultivates, observe the weeding damage of each blade.Bleach and wither from the blade of the tobacco that imports contrast DNA (the T-DNA zone of plasmid pBI121S).Contrast therewith is from the blade continuous growth of the tobacco that imports DNA of the present invention (A1) (plasmid pBI-NdG6-rSt-657, plasmid pBI-NdG6-rSt12-657, the T-DNA zone of pBI-NdG6-rSt-657F or pBI-NdG6-rSt12-657F).

Embodiment 20 imports plant with DNA of the present invention

Structure is used for importing with particle bombardment and agrobacterium co-cultivation the plasmid of DNA of the present invention (A2).At first, the DNA of the present invention (A2) that has nucleotide sequence shown in the SEQ ID NO:7 by pcr amplification.By with the genomic dna of actinomyces Ta Shi saccharopolyspora strain JCM 9383t as template with by the oligonucleotide that will form by nucleotide sequence shown in the SEQ ID NO:100 be used as primer by the oligonucleotide that nucleotide sequence shown in the SEQ ID NO:101 is formed and carry out PCR.Described PCR uses Expand HighFidelityPCR System (Boehringer Company).Keeping 97 ℃ after 2 minutes 1 time; Repeat 10 circulations, circulation comprise keep 97 ℃ 15 seconds, then 60 ℃ 30 seconds and then 72 ℃ 60 seconds; Carry out 15 circulations then, circulation comprise keep 97 ℃ 15 seconds, then 60 ℃ of 30 seconds and then 72 ℃ 1 minute (wherein each circulates in 72 ℃ maintenance increases by 20 seconds); Keep 72 ℃ to carry out PCR in 7 minutes then.The PCR product cloning zone of inserting pCR2.1-TOPO (Invitrogen Company) by the DNA with amplification produces plasmid pCR923Sp (Figure 31).Next, plasmid is imported escherichia coli jm109 competent cell (TakaraShuzo Company) and select the amicillin resistance cell.In addition, by using BigDye to stop the nucleotide sequence that cycle sequencing easy reaction test kit 2.0 editions (PE Applied Biosystems Company) and dna sequencing instrument 373S (PE Applied Biosystems Company) mensuration are included in the plasmid in the anti-penbritin bacterial strain.As a result, confirm that plasmid pCR923Sp has nucleotide sequence shown in the SEQ IDNO:7.

Digest the plasmid pKFrSt12 of design in embodiment 16 (3) to separate the DNA that comprises rSt12DNA of the present invention with restriction enzyme BamHI and SacI.Described DNA is inserted between the BglII restriction site of the pNdG6-Δ T that obtain among the embodiment 16 (2) and the SacI restriction site to obtain plasmid pNdG6-rSt12 (Figure 32).The DNA that comprises DNA of the present invention (A2) with restriction enzyme SphI and KpnI digested plasmid pCR923Sp with acquisition.With restriction enzyme SphI and KpnI digested plasmid pNdG6-rSt12 to remove 12 amino acid whose DNA of coding soybean (cv.Jack) RuBPC small subunit mature protein.In this position, the above-mentioned DNA that will comprise the DNA of the present invention (A2) that obtains from plasmid pCR923Sp inserts to obtain pSUM-NdG6-rSt-923 (Figure 33), wherein the CR16G6 promotor has been connected to the downstream of chimeric DNA, is right after after the sequence of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and does not have a variation of codon frame at DNA described in the described chimeric DNA.

Next, with restriction enzyme SphI digested plasmid pCR923Sp.After the DNA that obtains being equalled endization, described DNA is further digested the DNA that contains DNA of the present invention (A2) with separation with restriction enzyme KpnI with the KOD archaeal dna polymerase.Plasmid pKFrSt12 with restriction enzyme BspHI digestion generation in embodiment 16 (3).After the DNA that obtains being equalled endization, described DNA is further digested to remove the DNA of about 20bp with restriction enzyme KpnI with the KOD archaeal dna polymerase.At this place, to comprise the above-mentioned DNA that contains DNA of the present invention (A2) that obtains from plasmid pCR923Sp and insert obtaining to comprise the plasmid pKFrSt12-923 (Figure 34) of chimeric DNA, DNA of the present invention (A2) is right after after 12 amino acid whose nucleotide sequences of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and the mature protein thereafter of encoding and does not have a variation of codon frame in described chimeric DNA.Digest pKFrSt12-923 to obtain chimeric DNA with restriction enzyme SphI and KpnI, DNA wherein of the present invention (A2) is connected with 12 amino acid whose DNA of beginning of coding soybean (cv.Jack) RuBPC small subunit maturation protein.With restriction enzyme SphI and KpnI digested plasmid pNdG6-rSt12 to remove 12 amino acid whose DNA of coding soybean (cv.Jack) RuBPC small subunit maturation protein.In this position, to insert available from the above-mentioned chimeric DNA of plasmid pKFrSt12-923 to obtain plasmid pSUM-NdG6-rSt12-923 (Figure 35), wherein the CR16G6 promotor therefrom has been connected to the downstream of chimeric DNA, and the described DNA that comprises DNA of the present invention (A2) in described chimeric DNA is right after after 12 amino acid whose sequences of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and the mature protein thereafter of encoding and does not have a variation of codon frame.

Use the plasmid pSUM-NdG6-rSt-923 and the pSUM-NdG6-rSt12-923 that obtain, with particle bombardment DNA of the present invention (A2) is imported soybean with the program identical with embodiment 17 described methods.

Digest above-mentioned plasmid pSUM-NdG6-rSt-923 to separate the DNA that comprises chimeric DNA with restriction enzyme HindIII and EcoRI, the described DNA that comprises DNA of the present invention (A2) in described chimeric DNA is right after after the sequence of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and does not have a variation of codon frame.As in embodiment 18, producing pBI-NdG6-rSt657, will comprise above-mentioned DNA available from the chimeric DNA of plasmid pSUM-NdG6-rSt-923 and insert between the HindIII restriction site of binary vector pBI121S and the EcoRI restriction site to obtain pBI-NdG6-rSt-923 (Figure 36).In addition, digest above-mentioned plasmid pSUM-NdG6-rSt12-923 to separate the DNA that comprises chimeric DNA with HindIII and EcoRI, the described DNA that comprises DNA of the present invention (A2) in described chimeric DNA is right after after 12 amino acid whose sequences of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and the mature protein thereafter of encoding and does not have a variation of codon frame.To insert between the HindIII restriction site of binary vector pBI121S and the EcoRI restriction site to obtain pBI-NdG6-rSt12-923 (Figure 37) available from the chimeric DNA of pSUM-NdG6-rSt12-923.

With each the importing agrobacterium tumefaciens lba4404 among plasmid pBI-NdG6-rSt-923 and the pBI-NdG6-rSt12-923.The transformant cultivation that produces is being comprised 300 μ g/ml streptomycetes, in the LB substratum of 100 μ g/ml Rifampins and 25 μ g/ml kantlex.Select transformant to have the Agrobacterium bacterial strain of pBI-NdG6-rSt1-923 or pBI-NdG6-rSt12-923 with separation.

With the Agrobacterium bacterial strain with pBI-NdG6-rSt-923 and have in the Agrobacterium bacterial strain of pBI-NdG6-rSt12-923 each infect the blade of sterile culture tobacco.Under the program that is similar to the method for in embodiment 19, describing, obtain to have imported the tobacco of DNA of the present invention (A2).

From the transgene tobacco that obtains, get three (3) sheet leaves.Every leaf is divided into 4, and wherein every 5-7mm is wide.Plant every leaf on the MS nutrient agar that contains 0.1mg/L compound (II) and cultivation at room temperature in illumination.At the 7th day that cultivates, observe the weeding damage of each blade.Bleach and wither from the blade of the tobacco that imports contrast DNA (the T-DNA zone of plasmid pBI121S).Contrast therewith is from the blade continuous growth of the tobacco that imports DNA of the present invention (A2) (the T-DNA zone of plasmid pBI-NdG6-rSt-923 or plasmid pBI-NdG6-rSt12-923).

Embodiment 21 imports tobacco with DNA of the present invention (A3)

Structure imports DNA of the present invention (A3) with particle bombardment and agrobacterium co-cultivation the plasmid of plant.

At first, the DNA of the present invention (A3) that has nucleotide sequence shown in the SEQ ID NO:8 by pcr amplification.By with the genomic dna of the brick-red streptomycete ATCC 21469 of actinomyces as template with by the oligonucleotide that will form by nucleotide sequence shown in the SEQ ID NO:102 be used as primer by the oligonucleotide that nucleotide sequence shown in the SEQ ID NO:103 is formed and carry out PCR.Described PCR uses Expand High Fidelity PCR System (Boehringer Company).Keeping 97 ℃ after 2 minutes 1 time; Repeat 10 circulations, circulation comprise keep 97 ℃ 15 seconds, then 60 ℃ 30 seconds and then 72 ℃ 1 minute; Carry out 15 circulations then, circulation comprise keep 97 ℃ 15 seconds, then 60 ℃ of 30 seconds and then 72 ℃ 1 minute (wherein each circulates in 72 ℃ maintenance increases by 20 seconds); Keep 72 ℃ to carry out PCR in 7 minutes then.The PCR product cloning zone of inserting pCR2.1 (Invitrogen Company) by the DNA with amplification produces plasmid pCR671ET (Figure 38).In addition, the oligonucleotide of forming except the oligonucleotide that will be made up of nucleotide sequence shown in the SEQ ID NO:104 with by nucleotide sequence shown in the SEQ ID NO:103 obtains plasmid pCR671Bs (Figure 39) as the primer with the program that is similar to aforesaid method.Next, plasmid is imported escherichia coli jm109 competent cell (Takara Shuzo Company) and select the amicillin resistance cell.In addition, by using BigDye to stop the nucleotide sequence that cycle sequencing easy reaction test kit 2.0 editions (PE Applied Biosystems Company) and dna sequencing instrument 3100 (PE Applied Biosystems Company) mensuration are included in the plasmid in the anti-penbritin bacterial strain.As a result, confirm that plasmid pCR671ET and pCR671Bs have nucleotide sequence shown in the SEQ ID NO:8.

With restriction enzyme EcoT22I and KpnI digested plasmid pCR671ET to separate the DNA that comprises DNA of the present invention (A3).Will be described DNA insert between EcoT22I restriction site and the KpnI restriction site obtaining to comprise the plasmid pUCrSt671 (Figure 40) of chimeric DNA, DNA of the present invention (A3) is right after after the sequence of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and does not have a variation of codon frame in described chimeric DNA.With restriction enzyme NheI and KpnI digested plasmid pUCrSt671 to separate the DNA that comprises DNA of the present invention (A3).The plasmid pNdG6-rSt12 of acquisition in embodiment 16 (2) is to remove the DNA of about 80bp with restriction enzyme NheI and KpnI digestion.In this position, the above-mentioned DNA that comprises available from the DNA of the present invention (A3) of plasmid pUCrSt671 is inserted to obtain pSUM-NdG6-rSt-671 (Figure 41), wherein the CR16G6 promotor has been connected to the downstream of chimeric DNA, and DNA of the present invention (A3) is right after after the sequence of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and does not have a variation of codon frame in described chimeric DNA.

With restriction enzyme BspHI and KpnI digested plasmid pCR671Bs to separate the DNA that comprises DNA of the present invention (A3).Described DNA is inserted between the BspHI restriction site of the pKFrSt12 that obtain among the embodiment 16 (3) and the KpnI restriction site obtaining to comprise the plasmid pKFrSt12-671 (Figure 42) of chimeric DNA, and DNA wherein of the present invention (A3) is right after after 12 amino acid whose sequences of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and the mature protein thereafter of encoding and does not have a variation of codon frame.The plasmid pNdG6-rSt12 of acquisition in embodiment 20 is to remove the DNA of about 80bp with restriction enzyme NheI and KpnI digestion.At this place, insert and above-mentionedly comprise DNA available from the DNA of the present invention (A3) of plasmid pKFrSt12-671 to obtain pSUM-NdG6-rSt12-671 (Figure 43), wherein the CR16G6 promotor has been connected to the downstream of chimeric DNA, and DNA of the present invention (A3) is right after after 12 amino acid whose sequences of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and the mature protein thereafter of encoding and does not have a variation of codon frame in described chimeric DNA.

Use the plasmid pSUM-NdG6-rSt-671 and the pSUM-NdG6-rSt12-671 that obtain, with particle bombardment DNA of the present invention (A3) is imported soybean with the program identical with embodiment 17 described methods.

Digest above-mentioned plasmid pSUM-NdG6-rSt-671 separating chimeric DNA with restriction enzyme HindIII and EcoRI, DNA of the present invention (A3) is close to after the sequence of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and does not have a variation of codon frame in described chimeric DNA.To comprise above-mentioned DNA available from the chimeric DNA of plasmid pSUM-NdG6-rSt-671 inserts between the HindIII restriction site of the binary vector plasmid pBI121S that obtains among the embodiment 18 and the EcoRI restriction site to obtain pBI-NdG6-rSt-671 (Figure 44).In addition, digest above-mentioned plasmid pSUM-NdG6-rSt12-671 to separate the DNA that comprises chimeric DNA with restriction enzyme HindIII and EcoRI, the described DNA that comprises DNA of the present invention (A3) in described chimeric DNA is right after after 12 amino acid whose sequences of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and the mature protein thereafter of encoding and does not have a variation of codon frame.To insert between the HindIII restriction site of binary plasmid carrier pBI121S and the EcoRI restriction site to obtain pBI-NdG6-rSt12-671 (Figure 45) available from the chimeric DNA of plasmid pSUM-NdG6-rSt12-671.

With each the importing agrobacterium tumefaciens lba4404 among plasmid pBI-NdG6-rSt-671 and the pBI-NdG6-rSt12-671.The transformant cultivation that produces is being comprised 300 μ g/ml streptomycetes, in the LB substratum of 100 μ g/ml Rifampins and 25 μ g/ml kantlex.Select transformant to have pBI-NdG6-rSt-671 or pBI-NdG6-rSt12-671 agrobacterium strains with separation.

With the agrobacterium strains with pBI-NdG6-rSt-671 and have in the agrobacterium strains of pBI-NdG6-rSt12-671 each infect the blade of sterile culture tobacco.Under the program that is similar to the method for in embodiment 19, describing, obtain to have imported the tobacco of DNA of the present invention (A3).

From transgene tobacco, get three (3) sheet leaves.Every leaf is divided into 4, and wherein every 5-7mm is wide.Plant every leaf on the MS nutrient agar that contains 0.1mg/L compound (II) and cultivation at room temperature in illumination.At the 7th day that cultivates, observe the weeding damage of each blade.

Embodiment 22 expression of protein of the present invention (B1) in intestinal bacteria

(1) contains the generation of the transformed into escherichia coli of DNA of the present invention (B1)

By will in embodiment 3 (1), carrying out PCR as template by the chromosomal DNA from abortion within the first month of pregnancy look streptomycete IFO 12898 preparations.By adding the above-mentioned chromosomal DNA of 300ng, 4 μ l dNTP mixtures (mixture of every kind of 2.5mM of 4 kinds of dNTP), 5 μ l 10x ExTaq damping fluids, 0.5ExTaq polysaccharase (Takara Shuzo Company), distilled water and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:105 and the every kind of 200nM of oligonucleotide with nucleotide sequence shown in the SEQ ID NO:53, the PCR reaction soln amounts to 50 μ l.The reaction conditions of PCR is to remain on 97 ℃ after 2 minutes; Repeat 25 circulations, circulation comprise remain on 97 ℃ 15 seconds, then 60 ℃ 30 seconds, then 72 ℃ 90 seconds; Keep then 72 ℃ 4 minutes.Will the reaction soln after maintenance be connected with carrier pCR2.1-TOPO (Invitrogen company) and import intestinal bacteria TOP10F ' according to the incidental specification sheets of described carrier.Use QIAprep Spin Miniprep test kit (Qiagen company) from the intestinal bacteria transformant that obtains, to prepare plasmid DNA.Oligonucleotide that use is made up of nucleotide sequence shown in the SEQ IDNO:67 and the oligonucleotide be made up of nucleotide sequence shown in the SEQ ID NO:68 are as primer, according to the incidental specification sheets of described test kit, stop cycle sequencing FS easy reaction test kit (Applied Biosystems Japanese firm) with dyestuff and carry out sequencing reaction.Sequencing reaction uses the plasmid DNA that obtains as template.With dna sequencing instrument 373A (Applied Biosystems Japanese firm) analytical reaction product.Based on the result, will have the plasmid called after pCR657FD of nucleotide sequence shown in the SEQ IDNO:15.

Next, with restriction enzyme NdeI and HindIII digestion pCR657FD.Digestion product is carried out agarose gel electrophoresis.Cutting comprises the gel area of about 200bp DNA from gel.By use the QIA PhastGel to extract test kit (Qiagen company) purify DNA from the gel that reclaims according to incidental specification sheets.Connect the DNA and the plasmid pKSN2 of the acquisition that digests with NdeI and HindIII and import e. coli jm109 with connecting test kit version 1 (Takara Shuzo Company) according to the incidental specification sheets of described test kit.Prepare plasmid DNA from the intestinal bacteria transformant that obtains.Analyze its structure.Will be wherein between the NdeI site and HindIII site of about 200bp DNA insertion pKSN2 of code book invention protein (B1), comprise the plasmid called after pKSN657FD of nucleotide sequence shown in the SEQ ID NO:15.Plasmid pKSN657FD is imported e. coli jm109.With the intestinal bacteria transformant called after JM109/pKSN657FD that obtains.In addition, plasmid pKSN2 is imported e. coli jm109.The intestinal bacteria transformant called after JM109/pKSN2 that obtains.

(2) expression and the described recovery of protein of protein of the present invention (B1) in intestinal bacteria

With under each comfortable 37 ℃ of e. coli jm109/pKSN657FD and the e. coli jm109/pKSN2 at the 10ml TB substratum that comprises 50 μ g/ml penbritins (1.2% (w/v) Tryptones, 2.4% (w/v) yeast extract, 0.4% (w/v) glycerine, the 17mM potassium primary phosphate, the 72mM dipotassium hydrogen phosphate) middle overnight incubation.The media transfer that one milliliter (1ml) obtains is cultivated down to the 100ml TB substratum that comprises 50 μ g/ml penbritins and at 26 ℃.OD660 arrives after about 0.5 30 (30) minutes, with the final concentration that IPTG adds to 1mM, further cultivates 20 hours.

From each substratum, reclaim cell, wash and be suspended in the described damping fluid that 10ml comprises 1mM PMSF with 0.1M tris-HCl damping fluid (pH7.5).The cell suspension that obtains in work output 3, is carried out ultrasonoscope (Sonifer (Branson SonicPower Company)) 6 times under the condition of space factor 30%, each 3 minutes, so that obtain cell pyrolysis liquid.With cell pyrolysis liquid centrifugal (1,200xg, 5 minutes) back recovery supernatant liquor and centrifugal (150,000xg, 70 minutes) with reclaim the supernatant liquor fraction (below, the supernatant liquor fraction that will obtain from e. coli jm109/pKSN657FD is called " intestinal bacteria pKSN657FD extract ", and the supernatant liquor fraction that will obtain from e. coli jm109/pKSN2 is called " intestinal bacteria pKSN2 extract ").On 15%-25%SDS-PAGE, analyze a microlitre (1 μ l) above-mentioned supernatant liquor fraction and dye with CBB.As a result, detect in intestinal bacteria pKSN657FD extract than intestinal bacteria pKSN2 extract intensive band significantly more, it is positioned at the electrophoresis position corresponding to the 7kDa molecular weight.Show that e. coli jm109/pKSN657FD expresses protein of the present invention (B1).

(3) protein of the present invention (B1) is applied to compound (II) is converted into the reaction system of compound (III)

Prepare the reaction soln of 30 μ l and remain on 30 ℃ following 10 minutes.Reaction soln is made up of 0.1M potassium phosphate buffer (pH 7.0), and it comprises 3ppm and uses ¹⁴The compound of C mark (II), 2mM β-NADPH (hereinafter referred to as " component A ") (Oriental Yeast Company), the intestinal bacteria pKSN657FD extract that 9 μ l reclaim in embodiment 22 (2), the intestinal bacteria pKSN657F extract (hereinafter referred to as " component D ") that 0.1U/ml ferredoxin reductase (hereinafter referred to as " component C ") (Sigma Company) and 15 μ l reclaim in embodiment 4 (2) is formed.In addition, preparation wherein adds the reaction soln that ferredoxin (hereinafter referred to as " B component ") (Sigma Company) that 2mg/ml derives from spinach substitutes the reaction soln of intestinal bacteria pKSN657FD extract and do not add anything alternative intestinal bacteria pKSN657FD extract.Keep these reaction solns similarly.Three microlitres (3 μ l) 2N HCl and 90 μ l ethyl acetate are added and be mixed in the every kind of reaction soln that keeps later.8, under the 000xg that the reaction soln that produces is centrifugal to reclaim 75 μ l ethyl acetate layers.After the drying under reduced pressure ethyl acetate layer, resistates is dissolved in the 6.0 μ l ethyl acetate.(5.0 μ l) puts silica gel tlc plate (TLC silica-gel plate 60F with its five microlitre ₂₅₄20cmx 20cm, 0.25mm is thick, Merck company) on.Use chloroform, the mixture of the 6:1:2 of acetate and ethyl acetate launched the TLC plate about 1 hour.Allow solvent evaporation then.The TLC plate is exposed to imaging plate (Fuji Film Company) to spend the night.Then, go up the analysis imaging plate at Image Analyzer BAS2000 (Fuji FilmCompany).Inspection is corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).The result is displayed in Table 13.

Table 13

Embodiment 23 expression of protein of the present invention (B2) in intestinal bacteria

(1) contains the generation of the transformed into escherichia coli of DNA of the present invention (B2)

By will in embodiment 6 (1), carrying out PCR as template by the chromosomal DNA from Ta Shi saccharopolyspora strain JCM9383t preparation.By adding the above-mentioned chromosomal DNA of 300ng, 4 μ l dNTP mixtures (mixture of every kind of 2.5mM of 4 kinds of dNTP), 5 μ l 10x ExTaq damping fluids, 0.5ExTaq polysaccharase (Takara Shuzo Company), distilled water and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:106 and the every kind of 200nM of oligonucleotide with nucleotide sequence shown in the SEQ ID NO:63, the PCR reaction soln amounts to 50 μ l.The reaction conditions of PCR is to remain on 97 ℃ after 2 minutes; Repeat 25 circulations, circulation comprise remain on 97 ℃ 15 seconds, then 60 ℃ 30 seconds, then 72 ℃ 90 seconds; Keep then 72 ℃ 4 minutes.Will the reaction soln after maintenance be connected with carrier pCR2.1-TOPO (Invitrogen company) and import intestinal bacteria TOP10F ' according to the incidental specification sheets of described carrier.Use QIAprepSpin Miniprep test kit (Qiagen company) from the intestinal bacteria transformant that obtains, to prepare plasmid DNA.Oligonucleotide that use is made up of nucleotide sequence shown in the SEQ IDNO:67 and the oligonucleotide be made up of nucleotide sequence shown in the SEQ ID NO:68 are as primer, according to the incidental specification sheets of described test kit, stop cycle sequencing FS easy reaction test kit (Applied Biosystems Japanese firm) with dyestuff and carry out sequencing reaction.Sequencing reaction uses the plasmid DNA that obtains as template.With dna sequencing instrument 373A (Applied Biosystems Japanese firm) analytical reaction product.Based on the result, will have the plasmid called after pCR923FD of nucleotide sequence shown in the SEQ IDNO:16.

Next, with restriction enzyme NdeI and HindIII digested plasmid pCR923FD.Digestion product is carried out agarose gel electrophoresis.Cutting comprises the gel area of about 200bp DNA from gel.By use the QIA PhastGel to extract test kit (Qiagen company) purify DNA from the gel that reclaims according to incidental specification sheets.Connect the DNA and the plasmid pKSN2 of the acquisition that digests with NdeI and HindIII and import e. coli jm109 with connecting test kit version 1 (Takara Shuzo Company) according to the incidental specification sheets of described test kit.Prepare plasmid DNA from the intestinal bacteria transformant that obtains.Analyze its structure.Will be wherein between the NdeI site and HindIII site of about 200bp DNA insertion pKSN2 of code book invention protein (B2), comprise the plasmid called after pKSN923FD of nucleotide sequence shown in the SEQ ID NO:16.Plasmid pKSN923FD is imported e. coli jm109.With the intestinal bacteria transformant called after JM109/pKSN923FD that obtains.In addition, plasmid pKSN2 is imported e. coli jm109.The intestinal bacteria transformant called after JM109/pKSN2 that obtains.

(2) expression and the described recovery of protein of protein of the present invention (B2) in intestinal bacteria

With under each comfortable 37 ℃ of e. coli jm109/pKSN923FD and the e. coli jm109/pKSN2 at the 10ml TB substratum that comprises 50 μ g/ml penbritins (1.2% (w/v) Tryptones, 2.4% (w/v) yeast extract, 0.4% (w/v) glycerine, the 17mM potassium primary phosphate, the 72mM dipotassium hydrogen phosphate) middle overnight incubation.The media transfer that one milliliter (1ml) obtains is cultivated down to the 100ml TB substratum that comprises 50 μ g/ml penbritins and at 26 ℃.OD660 arrives after about 0.5 30 (30) minutes, with the final concentration that IPTG adds to 1mM, further cultivates 20 hours.

From each substratum, reclaim cell, wash and be suspended in the described damping fluid that 10ml comprises 1mMPMSF with 0.1M tris-HCl damping fluid (pH7.5).The cell suspension that obtains in work output 3, is carried out ultrasonoscope (Sonifer (Branson SonicPower Company)) 6 times under the condition of space factor 30%, each 3 minutes, so that obtain cell pyrolysis liquid.With cell pyrolysis liquid centrifugal (1,200xg, 5 minutes) back recovery supernatant liquor and centrifugal (150,000xg, 70 minutes) with reclaim the supernatant liquor fraction (below, the supernatant liquor fraction that will obtain from e. coli jm109/pKSN923FD is called " intestinal bacteria pKSN923FD extract ", and the supernatant liquor fraction that will obtain from e. coli jm109/pKSN2 is called " intestinal bacteria pKSN2 extract ").On 15%-25%SDS-PAGE, analyze a microlitre (1 μ l) above-mentioned supernatant liquor fraction and dye with CBB.By detecting in intestinal bacteria pKSN923FD extract corresponding to the electrophoresis position of 7kDa molecular weight, can confirm escherichia coli expression protein of the present invention (B2) than intestinal bacteria pKSN2 extract intensive band significantly more.

(3) protein of the present invention (B2) is applied to compound (II) is converted into the reaction system of compound (III)

Prepare the reaction soln of 30 μ l and remain on 30 ℃ following 10 minutes.Reaction soln is made up of 0.1M potassium phosphate buffer (pH 7.0), and it comprises 3ppm and uses ¹⁴The compound of C mark (II), 2mM β-NADPH (hereinafter referred to as " component A ") (Oriental Yeast Company), the intestinal bacteria pKSN923FD extract that 9 μ l reclaim in embodiment 23 (3), the intestinal bacteria pKSN657F extract (hereinafter referred to as " component D ") that 0.1U/ml ferredoxin reductase (hereinafter referred to as " component C ") (Sigma Company) and 15 μ l reclaim in embodiment 4 (2).In addition, preparation wherein adds ferredoxin (hereinafter referred to as " B component ") that 2mg/ml derives from spinach and (SigmaCompany) substitutes the reaction soln of intestinal bacteria pKSN923FD extract and do not add the reaction soln of anything alternative intestinal bacteria pKSN923FD extract.Keep these reaction solns similarly.Three microlitres (3 μ l) 2N HCl and 90 μ l ethyl acetate are added and be mixed in the every kind of reaction soln that keeps later.8, under the 000xg that the reaction soln that produces is centrifugal to reclaim 75 μ l ethyl acetate layers.After the drying under reduced pressure ethyl acetate layer, resistates is dissolved in the 6.0 μ l ethyl acetate.(5.0 μ l) puts silica gel tlc plate (TLC silica-gel plate 60F with its five microlitre ₂₅₄20cm x 20cm, 0.25mm is thick, Merck company) on.Use chloroform, the mixture of the 6:1:2 of acetate and ethyl acetate launched the TLC plate about 1 hour.Allow solvent evaporation then.The TLC plate is exposed to imaging plate (Fuji FilmCompany) to spend the night.Then, go up the analysis imaging plate at Image Analyzer BAS2000 (Fuji Film Company).Inspection is corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).By confirming that compound (III) is comprising component A, intestinal bacteria pKSN923FD extract, produce in the reaction of component C and component D, can confirm in the reaction system that compound (II) is converted into compound (III), can use protein of the present invention (B2) to replace deriving from the ferredoxin of spinach.

Embodiment 24 expression of protein of the present invention (B3) in intestinal bacteria

(1) has the generation of the transformed into escherichia coli of DNA of the present invention (B3)

Except will be in embodiment 11 (1) from the chromosomal DNA of brick-red streptomycete ATCC 21469 preparations as template and the oligonucleotide that will have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:107 and have nucleotide sequence shown in the SEQ ID NO:72 as the primer, be similar to embodiment 23 (1) described methods and carry out PCR.Be similar to embodiment 23 (1) described methods, use the reaction soln that obtains to obtain to have the plasmid pCR671FD of nucleotide sequence shown in the SEQ ID NO:17.

Next, utilize described plasmid, be similar to embodiment 23 (1) described methods and obtain wherein DNA of the present invention (B3) to be inserted the NdeI site of pKSN2 and the plasmid pKSN671FD between the HindIII site.By plasmid is imported e. coli jm109, can obtain to have e. coli jm109/pKSN671FD of DNA of the present invention (B3).

(2) expression and the described recovery of protein of protein of the present invention (B3) in intestinal bacteria

Use e. coli jm109/pKSN671FD, be similar to embodiment 23 (2) described methods and reclaim supernatant liquor fraction (hereinafter referred to as " intestinal bacteria pKSN671FD fraction ").On 15%-25%SDS-PAGE, analyze a microlitre (1 μ l) above-mentioned supernatant liquor fraction and dye with CBB.As a result, by detecting in intestinal bacteria pKSN671FD extract corresponding to the electrophoresis position of 7kDa molecular weight, can confirm escherichia coli expression protein of the present invention (B3) than intestinal bacteria pKSN2 extract intensive band significantly more.

(3) protein of the present invention (B3) is applied to compound (II) is converted into the reaction system of compound (III)

Except using the intestinal bacteria pKSN671FD extract that in embodiment 24 (2), reclaims, be similar to embodiment 23 (3) described methods and confirm corresponding to usefulness ¹⁴The spot of the compound of C mark (III) (Rf value 0.24 and 0.29).By confirming to comprise component A, intestinal bacteria pKSN671FD extract, produce compound (III) in the reaction of component C and component D, can confirm in the reaction system that compound (II) is converted into compound (III), can use protein of the present invention (B3) to replace deriving from the ferredoxin of spinach.

The preparation of embodiment 25 protein of the present invention (A4)

(1) preparation of cell crude extract

The freezing original seed that does not produce look streptomycete IFO 12735 is added 10ml A substratum (0.1% (w/v) glucose in the Boiling tube, 0.5% (w/v) Tryptones, 0.5% (w/v) yeast extract, 0.1% (w/v) dipotassium hydrogen phosphate, pH7.0) and 30 ℃ down vibration incubations 1 day to obtain pre-culture.Add eight milliliters (8ml) pre-culture in the 200mlA substratum and under 30 ℃, shook in the bottle rotational oscillation incubation 2 days at 500ml band baffle plate.Reclaim cell precipitation by the culture centrifugal (3,000xg, 10 minutes) that will produce.These cell precipitations are suspended in the 100mlB substratum (1% (w/v) glucose, 0.1% extractum carnis, 0.2% (w/v) tryptose) that comprises 100ppm compound (II) and under 30 ℃ in 500ml slope mouth flask reciprocating vibration incubation 20 hours.Reclaim cell precipitation by the culture centrifugal (3,000g, 10 minutes) that 2L is produced.The cell precipitation that produces with 1L0.1M potassium phosphate buffer (pH7.0) washing 2 times is to provide 136g cell precipitation.

With 1g cell precipitation 1ml-2ml these cell precipitations are suspended in the 0.1M potassium phosphate buffer (pH7.0), in cell suspension, add a mmole (1mM) PMSF, 5mM benzenyl amidine HCl, 1mMEDTA, 3 μ g/ml leupeptins, 3 μ g/ml pepstatins and 1mM two sulphur tritols.By using French press (1000kg/cm ²) (Ohtake Seisakusho) break repeatedly suspension 2 times obtains cell pyrolysis liquid.After cell pyrolysis liquid is centrifugal (40,000xg, 30 minutes), reclaim supernatant liquor and 150, under the 000xg centrifugal 1 hour to reclaim supernatant liquor (hereinafter referred to as " cell crude extract ").

The 30 μ l reaction solns that preparation is made up of 0.1M potassium phosphate buffer (pH7.0), it comprises 3ppm and uses ¹⁴The compound of C mark (II), 2.4mM β-NADPH (hereinafter referred to as " component A ") (Oriental Yeast Company), 0.5mg/ml derive from the ferredoxin (hereinafter referred to as " B component ") (Sigma Company) of spinach, the cell crude extract that 1U/ml ferredoxin reductase (hereinafter referred to as " component C ") (Sigma Company) and 15 μ l reclaim in embodiment 25 (1).With reaction soln remain on 30 ℃ following 1 hour.In addition, prepare similarly and keep not to add be used for above-mentioned reaction soln composition, be selected from component A, the reaction soln of at least a component of B component and component C.Three microlitres (3 μ l) 2N HCl and 90 μ l ethyl acetate are added and be mixed in the every kind of reaction soln that keeps later.8, under the 000xg that the reaction soln that produces is centrifugal to reclaim 75 μ l ethyl acetate layers.After the drying under reduced pressure ethyl acetate layer, resistates is dissolved in the 6.0 μ l ethyl acetate.(5.0 μ l) puts silica gel tlc plate (TLC silica-gel plate 60F with its five microlitre ₂₅₄20cmx 20cm, 0.25mm is thick, Merck company) on.Use chloroform, the mixture of the 6:1:2 of acetate and ethyl acetate launched the TLC plate about 1 hour.Allow solvent evaporation then.The TLC plate is exposed to imaging plate (Fuji Film Company) to spend the night.Then, go up the analysis imaging plate at Image Analyzer BAS2000 (Fuji FilmCompany).Inspection is corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).The result is displayed in Table 14.

Table 14

(3) fractional separation of cell crude extract

Ammonium sulfate is added in the saturated amount of cell crude extract to 45% that obtains among the embodiment 25 (1).After stirring under the ice-cooled condition, by 12, reclaimed supernatant liquor under the 000xg in centrifugal 30 minutes.In the saturated amount of the supernatant liquor to 55% that ammonium sulfate add is obtained with after stirring under the ice-cooled condition, by 12, reclaimed precipitation under the 000xg in centrifugal 10 minutes.With two three propane damping fluid (pH7.0) dissolution precipitations of the 20mM of 12.5ml.This solution is carried out PD10 post (AmershamPharmacia Company) and comprises proteinic fraction (hereinafter referred to as " 45-55% ammonium sulfate fraction ") with two three propane damping fluid (pH7.0) wash-outs of 20mM to reclaim 17.5ml.

(4) separation of protein of the present invention (A4)

The 45-55% ammonium sulfate fraction that will prepare in embodiment 25 (3) is expelled in the HiLoad26/10QSepharose HP post (Amersham Pharmacia Company).Next, after the two three propane damping fluids (pH 7.0) of the 20mM of 100ml flow to pillar, (gradient of NaCl is 0.004M/ minute with the NaCl of linear gradient, the NaCl range of concentrations is 0M-1M, and flow velocity is 4ml/ minute) the two three propane damping fluids of the 20mM that flows are recovered in the 30ml fraction of wash-out under the NaCl concentration of 0.12M-0.16M with classification.Further, the fraction that reclaims is carried out PD10 post (AmershamPhamacia Biotech Company) and comprise proteinic fraction with recovery with 20mM pair of three propane damping fluids (pH 7.0) wash-outs.

The fraction that reclaims is carried out PD10 post (Amersham Pharmacia Biotech Company),, comprise proteinic fraction so that reclaim with buffer A (comprise the 2mM potassium phosphate buffer of 1.5mMNaCl, pH 7.0) wash-out.Next, fraction is injected among the Bio-Scale Ceramic hydroxylapatite I type pillar CHT10-I (BioRad Company).20 milliliters of (20ml) buffer A are flow to pillar.Subsequently, the buffer B of buffer A and linear gradient (the 100mM potassium phosphate buffer that comprises 0.03mMNaCl; Linear gradient increased to 50% buffer B since 100% buffer A during 100 minutes, flow velocity is 2ml/ minute) flowing together is recovered in the fraction of wash-out under the concentration of buffer B of 4%-6% with classification.Further, the fraction that reclaims is carried out PD10 post (Amersham Pharmacia Biotech Company) and comprised proteinic fraction with 0.05M potassium phosphate buffer (pH7.0) wash-out with recovery.

The 0.05M potassium phosphate buffer (pH7.0) that comprises 2.0M ammonium sulfate of similar quantity is added and be mixed in the fraction of recovery.Then the fraction that reclaims is expelled in the 1ml RESOURSE PHE post (Amersham Pharmacia Biotech Cdmpany).Flow comprise the 5ml 0.05M potassium phosphate buffer (pH7.0) of 1M ammonium sulfate after, 0.05M (concentration gradient of ammonium sulfate is 0.1M/ minute to the ammonium sulfate of potassium phosphate buffer (pH 7.0) and linear gradient, the NaCl range of concentrations is 1M-0M, and flow velocity is 2ml/ minute) flowing together is recovered in the fraction of wash-out under the ammonium sulfate concentrations of about 0.4M-0.5M with classification.Analyzing the protein that in each fraction, comprises on the 10%-20% SDS-PAGE.

Be similar to embodiment 25 (2), replace the cell crude extract in embodiment 25 (2) the described reaction solns, at component A, B component, component C and usefulness ¹⁴Under the existence of the component of C mark (II), the fraction that adds and keep reclaiming.To keep later reaction soln TLC to analyze to check corresponding to usefulness ¹⁴The intensity of the spot of the compound of C mark (III).From gel, be recovered in the described protein that moves to about 45kDa position among the above-mentioned SDS-PAGE and use protein sequencer (AppliedBiosystems Company, Procise 494HT, the pulse liquid phase process) carry out amino acid sequence analysis so that the N-terminal aminoacid sequence is checked order.As a result, be provided at the aminoacid sequence of representing among the SEQ ID NO:113.

Embodiment 26 obtains DNA of the present invention (A4)

(1) do not produce the preparation of the chromosomal DNA of look streptomycete IFO 12735

At 50ml YEME substratum (0.3% (w/v) yeast extract, 0.5% (w/v) bactopeptone, 0.3% (w/v) wort, 1.0% (w/v) glucose, 34% (w/v) sucrose and 0.2% (v/v) 2.5MgCl ₂6H ₂O) will not produce look streptomycete IFO 12735 in 30 ℃ of following shaking culture 1 day to 3 days.Reclaim cell.With in the YEME substratum that comprises 1.4% (w/v) glycine and 60mM EDTA and the further vibration incubation 1 day of the cell suspension that obtains.From substratum, reclaim cell.With distilled water wash once after, with every 200mg cell 1ml with it be resuspended in damping fluid (100mM Tris-HCl (pH 8.0), 100mM EDTA, 10mMNaCl) in.The hen egg white lysozyme that adds every milliliter of two hectogamma (200 μ g/ml).Cell suspension was vibrated 1 hour down at 30 ℃.In addition, add 0.5%SDS and 1mg/ml Proteinase K.With cell suspension 55 ℃ of following incubations 3 hours.With phenol chloroform isoamyl alcohol extraction cell suspension 2 times to reclaim each water layer.Next, extract once to reclaim water layer with chloroform isoamyl alcohol.Obtain chromosomal DNA by the ethanol sedimentation water layer.

(2) do not produce the preparation of the chromosomal dna library of look streptomycete IFO 12735

With 3.2U restriction enzyme Sau3AI at 37 ℃ of following 38 micrograms (38 μ g) chromosomal DNAs of among embodiment 26 (1), preparing of digestion 60 minutes.Separate the Digestive system that obtains with 1% agarose gel electrophoresis.From gel, reclaim the DNA of about 2.0kbp.Extract test kit (Qiagen company) purify DNA and concentrated to obtain the solution that 20 μ l comprise target dna according to the incidental specification sheets of described test kit with the QIA PhastGel with ethanol sedimentation.With eight microlitres (8 μ l) dna solution, 100ng mixes from the solution I that is connected test kit second edition (Takara Shuzo Company) with 16 μ l with the plasmid vector pUC118 that handles with dephosphorylation with restriction enzyme BamHI digestion and kept 3 hours down at 16 ℃.Use to connect solution transformed into escherichia coli DH5 α, and be coated on the LB nutrient agar that comprises the 50mg/L penbritin with 37 ℃ of following overnight incubation.From nutrient agar, reclaim the bacterium colony that obtains.Extract plasmid.The plasmid that obtains is called chromosomal dna library.

(3) separation of DNA of the present invention (A4)

Carry out PCR by the chromosomal DNA that will in embodiment 26 (2), prepare as template.As primer, use oligonucleotide with nucleotide sequence shown in the SEQ ID NO:114 and pairing with oligonucleotide of nucleotide sequence shown in the SEQ ID NO:57.Based on nucleotide sequence shown in the design of aminoacid sequence shown in the SEQ ID NO:113 SEQ ID NO:114.Expand HiFiPCR system (Boehringer Manheim Company) is used for preparation feedback solution.By adding the above-mentioned chromosomal dna library of 2.5 μ l, 2 kinds every kind primer that amounts to 7.5pmol, 0.2 μ l dNTP mixture (mixture of every kind of 2mM of 4 kinds of dNTP), the 10x damping fluid of 0.2 μ l (comprises MgCl ₂), 0.38 μ l Expand HiFi enzyme mixture and distilled water, the PCR reaction soln amounts to 25 μ l.The reaction conditions of PCR is to remain on 97 ℃ after 2 minutes, repeating 10 circulations, circulation comprise remain on 97 ℃ 15 seconds, then 65 ℃ 30 seconds, then 72 ℃ 1 minute; Carry out 15 circulations then, circulation comprise remain on 97 ℃ 15 seconds, then 65 ℃ 30 seconds, then 72 ℃ 1 minute (wherein each circulation remains on 72 ℃ increases by 20 seconds); Remain on then 72 ℃ 7 minutes.After maintenance, 2.5 μ l reaction solns are used as template solution to carry out the PCR second time.As primer, use oligonucleotide with nucleotide sequence shown in the SEQ ID NO:115 and pairing with oligonucleotide of nucleotide sequence shown in the SEQ ID NO:57.Based on nucleotide sequence shown in the design of aminoacid sequence shown in the SEQ IDNO:113 SEQ ID NO:115.Be similar to aforesaid method, ExpandHiFi PCR system (Boehringer Manheim Company) is used for carrying out PCR.Reaction soln after keeping is carried out 2% agarose gel electrophoresis.Recovery comprises the gel area of the DNA of about 800bp.By use the QIA PhastGel to extract test kit (Qiagen company) purify DNA from the gel that reclaims according to incidental specification sheets.According to the incidental specification sheets of described carrier the DNA that obtains is connected to TA cloning vector pCRII-TOPO (Invitrogen company) and imports intestinal bacteria TOP10F '.Use Qiagen Tip20 (Qiagen company) from the intestinal bacteria transformant that obtains, to prepare plasmid DNA.According to the incidental specification sheets of described test kit, use has the primer of nucleotide sequence shown in the SEQ ID NO:67 and uses the primer with nucleotide sequence shown in the SEQ ID NO:68, stops cycle sequencing FS easy reaction test kit (Applied Biosystems Japanese firm) with Big Dye and carries out sequencing reaction.Sequencing reaction uses the plasmid that obtains as template.With dna sequencing instrument 3100 (Applied Biosystems Japanese firm) analytical reaction product.As a result, be provided at the nucleotide sequence shown in the Nucleotide 57 to 832 of the nucleotide sequence of representing among the SEQ ID NO:110.In the nucleotide sequence that provides, the amino acid 20 of aminoacid sequence shown in the coding of the Nucleotide 58-60 of nucleotide sequence shown in the SEQ ID NO:110 SEQ ID NO:113.

Next, under these conditions, the chromosomal dna library that will prepare in embodiment 26 (2) carries out PCR as template with Expand HiFi PCR system (Boehringer Manheim Company).As primer, use oligonucleotide with nucleotide sequence shown in the SEQ ID NO:116 and primer pairing with oligonucleotide of nucleotide sequence shown in the SEQ ID NO:59.With the dna clone of the amplification of about 1.4kb in cloning vector pCRII-TOPO.Utilize Qiagen Tip20 (Qiagen Company) from the intestinal bacteria transformant that obtains, to prepare plasmid DNA.According to the incidental specification sheets of described test kit, use has the primer of nucleotide sequence shown in the SEQ ID NO:67 and uses the primer with nucleotide sequence shown in the SEQ ID NO:68, stops cycle sequencing FS easy reaction test kit (Applied Biosystems Japanese firm) with Big Dye and carries out sequencing reaction.Sequencing reaction uses the plasmid that obtains as template.With dna sequencing instrument 3100 (Applied Biosystems Japanese firm) analytical reaction product.As a result, be provided at the nucleotide sequence shown in the Nucleotide 1 to 58 of the nucleotide sequence of representing among the SEQ ID NO:110.

Carry out from Nucleotide 3 ' the terminal clone who extends the DNA in downstream of Nucleotide 832 expressions of nucleotide sequence shown in the SEQ ID NO:110.Particularly, the 13 μ g that prepare in embodiment 26 (1) 37 ℃ of following digested overnight with 200U restriction enzyme HincII do not produce look streptomycete IFO 12735 chromosomal DNAs.Behind phenol extraction, by the ethanol sedimentation purify DNA.The DNA that obtains is used for producing the 20 μ l aqueous solution.With its four microlitre (4 μ l), the 15 μ M genome walking connectors (Genome Walker Adaptor) of 1.9 μ l, 1.6 μ l 10x connect damping fluid and the mixing of 0.5 μ l 6U/ μ l T4 ligase enzyme and keep spending the night under 16 ℃.Thereafter, keep 70 ℃ 5 minutes and add 72 μ l distilled water so that genome walking (Genome Walker) library to be provided.By PCR is carried out as template in described library.By primer AP1 (Universal Genome Walker test kit provides) that adds 1 μ l genome walking library and each total 200nM and oligonucleotide with nucleotide sequence shown in the SEQ ID NO:117, add 1 μ ldNTP mixture (mixture of every kind of 10mM of 4 kinds of dNTP), the 5x GC genome PCR damping fluid of 10 μ l, 2.2 μ l25mMMg (OAc) ₂, 10 μ l5M GC-Melt and 1 μ l Advantage-GC genome polysaccharase mixture and adding distilled water provide the PCR reaction soln that amounts to 50 μ l.The reaction conditions of PCR is to remain on 95 ℃ after 1 minute; Carry out 7 circulations, circulation comprise remain on 94 ℃ 10 seconds, then 72 ℃ 3 minutes; 36 circulations, circulation comprise keep 94 ℃ 10 seconds and then 68 ℃ 3 minutes; With keep 68 ℃ 7 minutes.50 times of reaction soln dilutions after will keeping with distilled water.This PCR product is called a PCR product and carries out another PCR as template.By primer AP2 (Universal Genome Walker test kit provides) that adds 1 μ l the one PCR product and each total 200nM and oligonucleotide with nucleotide sequence shown in the SEQ ID NO:118, add 1 μ l dNTP mixture (mixture of every kind of 10mM of 4 kinds of dNTP), the 5x GC genome PCR damping fluid of 10 μ l, 2.2 μ l 25mMMg (OAc) ₂, 10 μ l 5M GC-Melt and 1 μ l Advantage-GC genome polysaccharase mixture and adding distilled water provide the PCR reaction soln that amounts to 50 μ l.The reaction conditions of PCR is to remain on 95 ℃ after 1 minute; Carry out 5 circulations, circulation comprise remain on 94 ℃ 10 seconds, then 72 ℃ 3 minutes; 20 circulations, circulation comprise keep 94 ℃ 10 seconds and then 68 ℃ 3 minutes; With keep 68 ℃ 7 minutes.To keep later reaction soln to carry out 1% agarose gel electrophoresis.Recovery comprises the gel area of the DNA of about 1300bp.By use the QIA PhastGel to extract test kit (Qiagen company) purify DNA from the gel that reclaims according to incidental specification sheets.According to the incidental specification sheets of described carrier the DNA that obtains is connected to cloning vector pCRII-TOPO (Invitrogen company) and imports intestinal bacteria TOP10F '.Use QiagenTip20 (Qiagen company) from the intestinal bacteria transformant, to prepare plasmid DNA.According to the incidental specification sheets of described test kit, use oligonucleotide shown in the SEQ ID NO:67 and the oligonucleotide shown in the SEQ ID NO:68 as primer, stop cycle sequencing FS easy reaction test kit (Applied Biosystems Japanese firm) with Big Dye and carry out sequencing reaction.Sequencing reaction uses the plasmid that obtains as template.With dna sequencing instrument 3100 (Applied Biosystems Japanese firm) analytical reaction product.As a result, be provided at the nucleotide sequence shown in the Nucleotide 644 to 1454 of the nucleotide sequence of representing among the SEQ ID NO:110.Result as the Nucleotide with all analyses connects is provided at the nucleotide sequence of representing among the SEQ ID NO:110.In described nucleotide sequence, there are two open reading-frame (ORF)s (ORF).Therefore, comprise by 1236 Nucleotide (comprising terminator codon) and form and the nucleotide sequence (SEQ ID NO:109) of 411 amino-acid residues of encoding (SEQ ID NO:108) and form and the nucleotide sequence (SEQ ID NO:112) of 63 amino-acid residues (the SEQID NO:111) that encode by 192 Nucleotide (comprising terminator codon).The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ IDNO:109 (SEQ ID NO:108) it is calculated that and is 45465Da.In addition, comprise the aminoacid sequence of being measured from the N-terminal amino acid sequencing of protein of the present invention (A4) (SEQ ID NO:113) by described nucleotide sequence coded aminoacid sequence.The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:112 (SEQ ID NO:111) it is calculated that and is 6871Da.

Embodiment 27 expression of protein of the present invention (A4) in intestinal bacteria

(1) has the generation of the transformed into escherichia coli of DNA of the present invention (A4)

Carry out PCR by the chromosomal DNA that will in embodiment 26 (1), never produce look streptomycete IFO 12735 preparations as template with by use Expand HiFi PCR System (Boehringer ManheimCompany).As primer, use oligonucleotide and have the pairing (hereinafter referred to as " primer is to 25 ") of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:120 or have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:119 and have the pairing (hereinafter referred to as " primer is to 26 ") of the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:121 with nucleotide sequence shown in the SEQ ID NO:119.By adding 2 kinds every kind primer that amounts to 300nM, the above-mentioned chromosomal DNA of 50ng, 5.0 μ ldNTP mixtures (mixture of every kind of 2.0mM of 4 kinds of dNTP), 5.0 μ l 10x Expand HF damping fluids (comprise MgCl ₂) and 0.75 μ l Expand HiFi enzyme mixture and distilled water, the PCR reaction soln amounts to 50 μ l.The reaction conditions of PCR is to remain on 97 ℃ after 2 minutes; Repeat 10 circulations, circulation comprise remain on 97 ℃ 15 seconds, then 60 ℃ 30 seconds, then 72 ℃ 1 minute; Carry out 15 circulations then, circulation comprise remain on 97 ℃ 15 seconds, then 60 ℃ 30 seconds, then 72 ℃ 1 minute (wherein each circulation increase by 20 seconds remain on 72 ℃); Remain on then 72 ℃ 7 minutes.After maintenance, reaction soln is carried out 1% agarose gel electrophoresis.Use primer the gel of 25 reaction soln to be reclaimed the gel area of the DNA that comprises about 1.3kbp from experience.Use the gel area that reclaims the DNA that comprises about 1.6kbp the gel of primer to 26 reaction soln from experience.By using the QIA PhastGel to extract test kit (Qiagen company) purify DNA from the gel of each recovery according to incidental specification sheets.According to the incidental specification sheets of described carrier the DNA that obtains is connected to cloning vector pCRII-TOPO (Invitrogen company) and imports intestinal bacteria TOP10F '.Use Qiagen Tip20 (Qiagen company) from the intestinal bacteria transformant that obtains, to prepare plasmid DNA.Next, according to the incidental specification sheets of described test kit, use SEQ ID NO:67, SEQ ID NO:68, oligonucleotide effect primer shown in SEQ ID NO:122 and the SEQ ID NO:123 stops cycle sequencing FS easy reaction test kit (Applied Biosystems Japanese firm) with Big Dye and carries out sequencing reaction.Sequencing reaction uses the plasmid DNA that obtains as template.With dna sequencing instrument 3100 (Applied Biosystems Japanese firm) analytical reaction product.Based on the result, will have the plasmid called after pCR646 of nucleotide sequence shown in the SEQ ID NO:109 and will have the plasmid called after pCR646F of nucleotide sequence shown in the SEQ ID NO:110.

Next, each among usefulness restriction enzyme NdeI and HindIII digested plasmid pCR646 and the pCR646F.Digestion product is carried out agarose gel electrophoresis.Cutting comprises the gel area of about 1.3kbp DNA from the gel that carries out the pCR646 digestion product.Cutting comprises the gel area of about 1.6kbp DNA from the gel that carries out the pCR646F digestion product.By using the QIA PhastGel to extract test kit (Qiagen company) purify DNA from the gel of each recovery according to incidental specification sheets.Connect the DNA and the plasmid pKSN2 of the every kind of acquisition that digests with NdeI and HindIII and import e. coli jm109 with connecting test kit version 1 (Takara ShuzoCompany) according to the incidental specification sheets of described test kit.Prepare plasmid DNA from the intestinal bacteria transformant that obtains.Analyze its structure.Will be wherein between the NdeI site and HindIII site of about 1.3kbp DNA insertion pKSN2 of code book invention protein (A4), comprise the plasmid called after pKSN646 of nucleotide sequence shown in the SEQ ID NO:109.In addition, will be wherein between the NdeI site and HindIII site of about 1.6kbp DNA insertion pKSN2 of code book invention protein (A4), comprise the plasmid called after pKSN646F of nucleotide sequence shown in the SEQ ID NO:110.With each the importing e. coli jm109 among above-mentioned plasmid pKSN646 and the pKSN646F.With intestinal bacteria transformant difference called after JM109/pKSN646 and the JM109/pKSN646F that obtains.In addition, plasmid pKSN2 is imported e. coli jm109.The intestinal bacteria transformant called after JM109/pKSN2 that obtains.

(2) expression and the described recovery of protein of protein of the present invention (A4) in intestinal bacteria

With e. coli jm109/pKSN646, under each comfortable 37 ℃ of JM109/pKSN646F and the JM109/pKSN2 at the 10ml TB substratum that comprises 50 μ g/ml penbritins (1.2% (w/v) Tryptones, 2.4% (w/v) yeast extract, 0.4% (w/v) glycerine, the 17mM potassium primary phosphate, the 72mM dipotassium hydrogen phosphate) middle overnight incubation.The media transfer that one milliliter (1ml) obtains is cultivated down to the 100ml TB substratum that comprises 50 μ g/ml penbritins and at 26 ℃.When OD660 arrives approximately 0.5 the time, the 5-amino-laevulic acid is added to the final concentration of 500 μ M, continue to cultivate.After 30 (30) minutes,, further cultivated 17 hours the final concentration that IPTG adds to 1mM.

From each substratum, reclaim cell, wash and be suspended in the above-mentioned damping fluid that 10ml comprises 1mMPMSF with 0.1M tris-HCl damping fluid (pH 7.5).The cell suspension that obtains in work output 3, is carried out ultrasonoscope (Sonifer (Branson SonicPower Company)) 6 times under the condition of space factor 30%, each 3 minutes, so that obtain cell pyrolysis liquid.With cell pyrolysis liquid centrifugal (1,200xg, 5 minutes) back recovery supernatant liquor and centrifugal (150,000xg, 70 minutes) with reclaim the supernatant liquor fraction (below, the supernatant liquor fraction that will obtain from e. coli jm109/pKSN646 is called " intestinal bacteria pKSN646 extract ", the supernatant liquor fraction that will obtain from e. coli jm109/pKSN646F is called " intestinal bacteria pKSN646F extract ", and the supernatant liquor fraction that will obtain from e. coli jm109/pKSN2 is called " intestinal bacteria pKSN2 extract ").On 15%-25%SDS-PAGE, analyze a microlitre (1 μ l) above-mentioned supernatant liquor fraction and dye with CBB.The result, by in intestinal bacteria pKSN646 extract and intestinal bacteria pKSN646F extract, all detecting than intestinal bacteria pKSN2 extract intensive band significantly more, the electrophoresis position that it is positioned at corresponding to the 45kDa molecular weight can confirm that protein of the present invention (A4) is at expression in escherichia coli.

Prepare the reaction soln of 30 μ l and remain on 30 ℃ following 10 minutes.Reaction soln is made up of 0.1M potassium phosphate buffer (pH 7.0), and it comprises 3ppm and uses ¹⁴The compound of C mark (II), 2mM β-NADPH (hereinafter referred to as " component A ") (Oriental Yeast Company), 2mg/ml derives from the ferredoxin (hereinafter referred to as " B component ") (Sigma Company) of spinach, the supernatant liquor fraction that 0.1U/ml ferredoxin reductase (hereinafter referred to as " component C ") (Sigma Company) and 18 μ l reclaim in embodiment 27 (2).In addition, prepare similarly and keep not to add be used for above-mentioned reaction soln composition, be selected from component A, the reaction soln of at least a component of B component and component C.Three microlitres (3 μ l) 2N HCl and 90 μ l ethyl acetate are added and be mixed in the every kind of reaction soln that keeps later.8, under the 000xg that the reaction soln that produces is centrifugal to reclaim 75 μ l ethyl acetate layers.After the drying under reduced pressure ethyl acetate layer, resistates is dissolved in the 6.0 μ l ethyl acetate.(5.0 μ l) puts silica gel tlc plate (TLC silica-gel plate 60F with its five microlitre ₂₅₄20cm x 20cm, 0.25mm is thick, Merck company) on.Use chloroform, the mixture of the 6:1:2 of acetate and ethyl acetate launched the TLC plate about 1 hour.Allow solvent evaporation then.The TLC plate is exposed to imaging plate (Fuji Film Company) to spend the night.Then, go up the analysis imaging plate at Image Analyzer BAS2000 (FujiFilm Company).Inspection is corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).Can confirm that compound (III) is comprising component A, B component in the reaction soln of component C and intestinal bacteria pKSN646 extract, or is comprising component A, B component, the production in the reaction soln of component C and intestinal bacteria pKSN646F.

The sequence identity that embodiment 28 is relevant with protein of the present invention

By using GENETYX-WIN the 5th edition (Software Development Company) to analyze the sequence identity relevant with DNA of the present invention with protein of the present invention.Produce contrast by carrying out homology analysis with Lipman-Pearson method (Lipman, D.J. and Pearson, W.R., Science, 227,1435-1441, (1985)).

About the aminoacid sequence of protein of the present invention (A1) to (A4), determine each other and the sequence identity of the known protein matter of highest homology.The result represents in table 15.

Table 15

	Protein of the present invention (A1)	Protein of the present invention (A2)	Protein of the present invention (A3)	Protein of the present invention (A4)	The known protein matter * of highest homology
	Protein of the present invention (A1)	Protein of the present invention (A2)	Protein of the present invention (A3)	Protein of the present invention (A4)	The known protein matter * of highest homology	Protein of the present invention (A1)	100％	47％	64％	48％	73％ AAC25766
Protein of the present invention (A2)	47％	100％	48％	51％	52％ CAB46536	Protein of the present invention (A1)	100％	47％	64％	48％	73％ AAC25766
Protein of the present invention (A2)	47％	100％	48％	51％	52％ CAB46536	Protein of the present invention (A3)	64％	48％	100％	46％	67％ AAC25766
Protein of the present invention (A4)	48％	51％	46％	100％	50％ CAB46536	Protein of the present invention (A3)	64％	48％	100％	46％	67％ AAC25766

* sequence identity shows at the top, and the registration number of the protein that provides in Entrez database (information center provides by biotechnology, http://www3.ncbi.nlm.nih.gov/Entrez/) shows in the bottom.

Nucleotide sequence about DNA of the present invention (A1) with nucleotide sequence shown in the SEQ ID NO:6, nucleotide sequence with DNA of the present invention (A2) of nucleotide sequence shown in the SEQ ID NO:7, have nucleotide sequence shown in the SEQ ID NO:8 DNA of the present invention (A3) nucleotide sequence and have the nucleotide sequence of the DNA of the present invention (A4) of nucleotide sequence shown in the SEQ ID NO:109, determine each other and to the sequence identity of the known of highest homology.The result represents in table 16.

Table 16

SEQ ID NO:6 [the present invention

SEQ ID NO:7 [the present invention

SEQ ID NO:8 [the present invention

SEQ ID NO:109 [the present invention

The known * of highest homology

	DNA(A1)]	DNA(A2)]	DNA(A3)]	DNA(A4)]
	DNA(A1)]	DNA(A2)]	DNA(A3)]	DNA(A4)]		SEQ ID NO:6 [DNA of the present invention (A1)]	100％	61％	74％	62％	77％ AF072709
SEQ ID NO:7 [DNA of the present invention (A2)]	61％	100％	64％	65％	66％ Y18574	SEQ ID NO:6 [DNA of the present invention (A1)]	100％	61％	74％	62％	77％ AF072709
SEQ ID NO:7 [DNA of the present invention (A2)]	61％	100％	64％	65％	66％ Y18574	SEQ ID NO:8 [DNA of the present invention (A3)]	74％	64％	100％	63％	75％ AF072709
SEQ ID NO:109 [DNA of the present invention (A4)]	62％	65％	63％	100％	64％ Y18574	SEQ ID NO:8 [DNA of the present invention (A3)]	74％	64％	100％	63％	75％ AF072709

* sequence identity shows at the top, and the registration number of the gene that provides in Entrez database (information center provides by biotechnology, http://www3.ncbi.nlm.nih.gov/Entrez/) shows in the bottom.

About the aminoacid sequence of protein of the present invention (B1) to (B4), determine each other and the sequence identity of the known protein matter of highest homology.The result represents in table 17.

Table 17

	Protein of the present invention (B1)	Protein of the present invention (B2)	Protein of the present invention (B3)	Protein of the present invention (B4)	The known protein matter * of highest homology
	Protein of the present invention (B1)	Protein of the present invention (B2)	Protein of the present invention (B3)	Protein of the present invention (B4)	The known protein matter * of highest homology	Protein of the present invention (B1)	100％	45％	78％	41％	76％ AAC25765

Protein of the present invention (B2)	45％	100％	40％	41％	60％ AAF71770
Protein of the present invention (B2)	45％	100％	40％	41％	60％ AAF71770	Protein of the present invention (B3)	78％	40％	100％	40％	73％ AAC25765
Protein of the present invention (B4)	41％	41％	40％	100％	55％ AAA26824	Protein of the present invention (B3)	78％	40％	100％	40％	73％ AAC25765

Nucleotide sequence about DNA of the present invention (B1) with nucleotide sequence shown in the SEQ ID NO:15, nucleotide sequence with DNA of the present invention (B2) of nucleotide sequence shown in the SEQ ID NO:16, have nucleotide sequence shown in the SEQ ID NO:17 DNA of the present invention (B3) nucleotide sequence and have the nucleotide sequence of the DNA of the present invention (B4) of nucleotide sequence shown in the SEQ ID NO:112, determine each other and to the sequence identity of the known of highest homology.The result represents in table 18.

Table 18

	SEQ ID NO:15 [DNA of the present invention (B1)]	SEQ ID NO:16 [DNA of the present invention (B2)]	SEQ ID NO:17 [DNA of the present invention (B3)]	SEQ ID NO:112 [DNA of the present invention (B4)]	The known * of highest homology
	SEQ ID NO:15 [DNA of the present invention (B1)]	SEQ ID NO:16 [DNA of the present invention (B2)]	SEQ ID NO:17 [DNA of the present invention (B3)]	SEQ ID NO:112 [DNA of the present invention (B4)]	The known * of highest homology	SEQ ID NO:15 [DNA of the present invention (B1)]	100％	60％	80％	59％	84％ AF072709
SEQ ID NO:16 [DNA of the present invention (B2)]	60％	100％	60％	59％	66％ M32238	SEQ ID NO:15 [DNA of the present invention (B1)]	100％	60％	80％	59％	84％ AF072709
SEQ ID NO:16 [DNA of the present invention (B2)]	60％	100％	60％	59％	66％ M32238	SEQ ID NO：	80％	60％	100％	65％	79％

17 [DNA of the present invention (B3)]					AF072709
17 [DNA of the present invention (B3)]					AF072709	SEQ ID NO:112 [DNA of the present invention (B4)]	59％	59％	65％	100％	66％ M32239

Embodiment 29 uses the PCR of the oligonucleotide of the partial nucleotide sequence with DNA of the present invention (A) as primer

Chromosomal DNA by the abortion within the first month of pregnancy look streptomycete IFO 12898 that will in embodiment 2, prepare; The chromosomal DNA of the Ta Shi saccharopolyspora strain JCM 9383t of preparation in embodiment 5; The chromosomal DNA of the light gray streptomycete ATCC 11796 of preparation in embodiment 9; The chromosomal DNA of the brick-red streptomycete ATCC 21469 of preparation in embodiment 11; In embodiment 26 in the chromosomal DNA that does not produce look streptomycete IFO 12735 of preparation each; With the chromosomal DNA of the grey brown streptomycete IFO 12870t that is similar to embodiment 2 described method preparations, each among hot lightskyblue streptomycete IFO14273t and the black walnut streptomycete IFO 13445 is carried out PCR as template.As primer, use 5 pairs of primers shown in the table 19.Be displayed in Table 19 by the prediction size of use based on the DNA of the right pcr amplification of each primer of the nucleotide sequence of SEQ ID NO:6.

Table 19

Primer is right	Primer	Primer	The DNA of amplification
Primer is right	Primer	Primer	The DNA of amplification	14	SEQ ID NO：124	SEQ ID NO：129	About 800bp
15	SEQ ID NO：125	SEQ ID NO：129	About 600bp	14	SEQ ID NO：124	SEQ ID NO：129	About 800bp
15	SEQ ID NO：125	SEQ ID NO：129	About 600bp	16	SEQ ID NO：126	SEQ ID NO：129	About 600bp
17	SEQ ID NO：127	SEQ ID NO：129	About 580bp	16	SEQ ID NO：126	SEQ ID NO：129	About 600bp
17	SEQ ID NO：127	SEQ ID NO：129	About 580bp	18	SEQ ID NO：128	SEQ ID NO：129	About 580bp

By adding in 2 primers of pairing shown in the 200nM table 19 each, add the 10ng chromosomal DNA, 0.5 μ l dNTP mixture (mixture of every kind of 10mM of 4 kinds of dNTP), 5 μ l 5xGC genome PCR damping fluids, 1.1 μ l 25mM Mg (OAc) ₂, 5 μ l 5M GC-Melt and 0.5 μ lAdvantage-GC genome polysaccharase mixture and adding distil water, the PCR reaction soln amounts to 25 μ l.Reaction conditions be remain on 95 ℃ 1 minute; Repeat 30 circulations, circulation comprise remain on 94 ℃ 15 seconds, then 60 ℃ 30 seconds, then 72 ℃ 1 minute; With remain on 72 ℃ 5 minutes.With every kind of reaction soln after the 3% agarose gel electrophoresis analysis maintenance.The result shows in Figure 46 and in table 20 and the table 21.Using primer to 14,15, in 16,17 and 18 in each or the whole situation and using the amplification of from the situation of chromosomal DNA as template of arbitrary bacterial strain preparation, observing the big or small DNA of prediction.

Table 20

* the DNA of "+" expression prediction size is detected, and "-" expression does not detect.

Table 21

Embodiment 30 uses DNA that partial nucleotide sequence and the DNA of the present invention (A) by DNA of the present invention (A) the form hybridization as probe

(1) probe preparation

As produce the DNA that forms by the partial nucleotide sequence of the partial nucleotide sequence of DNA of the present invention (A1) or DNA of the present invention (A1) with the probe (probe of DIG mark) of digoxigenin mark.Chromosomal DNA by the abortion within the first month of pregnancy look streptomycete IFO 12898 that will prepare in embodiment 3 is used as primer as template with by the oligonucleotide that will be made up of nucleotide sequence shown in the SEQ ID NO:93 with by the oligonucleotide that nucleotide sequence shown in the SEQ ID NO:94 is formed, and carries out PCR according to incidental handbook use PCR DIG probe synthetic agent box (Roche Diagnostics GmbH Company).Each amounts to 200nM by adding 2 kinds of primers, add the 50ng chromosomal DNA, 2.5 μ l dNTP mixture (mixture of every kind of 2.0mM of 4 kinds of dNTP), 2.5 μ l PCR DIG mixture (using the mixture of every kind of 2.0mM of 4 kinds of dNTP of DIG mark), 5 μ l 10xPCR damping fluids and 0.75 μ l Expand HiFi enzyme mixture and adding distil water, the PCR reaction soln amounts to 50 μ l.Reaction conditions is to remain on 95 ℃ after 2 minutes; Repeat 10 circulations, circulation comprise remain on 95 ℃ 10 seconds, then 60 ℃ 30 seconds, then 72 ℃ 2 minutes; Carry out 15 circulations then, circulation comprise remain on 95 ℃ 10 seconds, then 60 ℃ 30 seconds, then 72 ℃ 2 minutes (wherein each circulation remains on 72 ℃ increases by 20 seconds); Remain on then 72 ℃ 7 minutes.Reaction soln after keeping is carried out 1% agarose gel electrophoresis.As a result, confirm the amplification of the DNA of about 1.3kb.The DNA that reclaims amplification is to obtain with digoxigenin mark and the DNA with nucleotide sequence shown in the SEQ ID NO:6.Under similar approach, by with the chromosomal DNA of abortion within the first month of pregnancy look streptomycete IFO12898 as template with by the oligonucleotide that will form by nucleotide sequence shown in the SEQ ID NO:130 be used as primer by the oligonucleotide that nucleotide sequence shown in the SEQ ID NO:131 is formed and carry out PCR.Will the DNA by described pcr amplification reclaim DNA with the nucleotide sequence that obtains to represent with digoxigenin mark and Nucleotide 57-730 with nucleotide sequence shown in the SEQ ID NO:6.

Under similar approach, the chromosomal DNA by the Ta Shi saccharopolyspora strain JCM 9393t that will prepare in embodiment 6 is as template with by the oligonucleotide that will be made up of nucleotide sequence shown in the SEQ ID NO:61 be used as primer by the oligonucleotide that nucleotide sequence shown in the SEQ ID NO:62 is formed and carry out PCR.The DNA of recovery by described pcr amplification is to obtain with the digoxigenin mark and to have the DNA of nucleotide sequence shown in the SEQ ID NO:7.

In addition, under similar method, the chromosomal DNA by the brick-red streptomycete ATCC 21469 that will prepare in embodiment 11 is as template with by the oligonucleotide that will be made up of nucleotide sequence shown in the SEQ ID NO:70 be used as primer by the oligonucleotide that nucleotide sequence shown in the SEQ ID NO:71 is formed and carry out PCR.The DNA of recovery by described pcr amplification is to obtain with the digoxigenin mark and to have the DNA of nucleotide sequence shown in the SEQ ID NO:8.In addition, by with above-mentioned chromosomal DNA as template with by the oligonucleotide that will form by nucleotide sequence shown in the SEQ ID NO:132 be used as primer by the oligonucleotide that nucleotide sequence shown in the SEQ ID NO:133 is formed and carry out PCR.The DNA of recovery by described pcr amplification is with the DNA of the nucleotide sequence that obtains to represent with digoxigenin mark and the Nucleotide 21-691 with nucleotide sequence shown in the SEQ ID NO:8.

(2) dot hybridization

The DNA (DNA that comprises DNA of the present invention (A1)) of the pKSN657 that will in embodiment 4, prepare, the DNA (DNA that comprises DNA of the present invention (A2)) of the pKSN923 of preparation in embodiment 7, the DNA (DNA that comprises DNA of the present invention (A3)) of pKSN671 of preparation in embodiment 12, in embodiment 14 DNA (DNA that comprises DNA of the present invention (A9)) of the pKSNSCA of preparation and in embodiment 10 each among the DNA (DNA that comprises DNA of the present invention (A10)) of the pKSN11796 of preparation with the amount trace of 100ng and 10ng to nylon membrane HybondN ⁺On (Amersham Pharmacia Company).The film that UV-light point to is obtained with transilluminator 5 minutes.

According to incidental handbook DIG-High Prime dna marker and detection Starter test kit II (Roche Diagnostics GmbH Company) are used for hybridization and detection.As probe, use each among the DNA that uses the digoxigenin mark and in embodiment 30 (1), produce, it kept 5 minutes and cooling (hereinafter referred to as " probe of DIG mark ") in ice fast then at 100 ℃.The above-mentioned film of trace was being vibrated 30 minutes in the 2.0ml DIGEasyHyb that described test kit provides under 42 ℃.Next, with 2.0ml Dig Easy Hyb, the probe of 5.0 μ l DIG marks and film phonograph seal in the plastics bag with hybridization, and remain on 42 ℃ following 18 hours.Reclamation film at room temperature vibrated 2 times 5 minutes in comprising the 2x SSC of 0.1%SDS, vibrated 2 times 15 minutes in the 0.5x SSC that is comprising 0.1%SDS under 65 ℃ then.Subsequently, film was vibrated 2 minutes in the 50ml lavation buffer solution, at room temperature in the 50ml confining liquid, vibrated 30 minutes then, in the 2.0ml antibody-solutions, vibrated 30 minutes then, in the 50ml lavation buffer solution, vibrated 2 times 15 minutes then.In addition, vibration also kept film phonograph seal at room temperature 18 hours in the hybridization bag that contains the 2.0ml chromogenic substrate after 5 minutes in 50ml detection damping fluid.Using 10ng and 100ng pKSN657, pKSN923, pKSN671 detects signal in every kind of situation that the every kind of reagent of each is hybridized among pKSNSCA and the pKSN11796.

Embodiment 31 obtains DNA of the present invention (A11)

(1) preparation of the chromosomal DNA of black walnut streptomycete IFO 13445

At 50ml YGY substratum (0.5% (w/v) yeast extract, 0.5% (w/v) Tryptones, 0.1% (w/v) glucose and 0.1% (w/v) K ₂HPO ₄, pH 7.0) in black walnut streptomycete IFO 13445 30 ℃ of following shaking culture 3 days.Reclaim cell.With in the YGY substratum that comprises 1.4% (w/v) glycine and 60mM EDTA and the further vibration incubation 1 day of the cell suspension that obtains.From substratum, reclaim cell.With distilled water wash once after, it is suspended in the 3.5ml buffer B 1 (50mM Tris-HCl (pH 8.0), 50mM EDTA, 0.5% tween 20 and 0.5%Triton X-100).The lysozyme soln of 80 microlitres (80 μ l), 100 μ g/ml and 100 μ l Qiagen proteolytic enzyme (600mAU/ml, Qiagen Company) are added suspension and remain on 37 ℃ 1 hour.Next, add 1.2ml buffer B 2 (3M Guanidinium hydrochloride and 20% tween 20s), mix and keep 50 ℃ 30 minutes.The cell pyrolysis liquid that obtains is added the Qiagen genome chip 100G (Qiagen Company) that is equilibrated among the damping fluid QBT (750mM NaCl, 50mM MOPS (pH7.0), 15% Virahol and 0.15% TritonX-100).Next, after with 7.5ml damping fluid QC (50mM MOPS (pH7.0) and 15% Virahol) washing chip 2 times, by 5ml damping fluid QF (1.25M NaCl, 50mM Tris HCl (pH 8.5), the 15% Virahol) eluted dna that flows.Three and 5/10ths milliliters of (3.5ml) Virahols are mixed in the dna solution of acquisition with precipitation and reclaim chromosomal DNA.After with 70% washing with alcohol, the DNA that reclaims is dissolved in the 1ml TB damping fluid.

(2) has the separation of the DNA of DNA of the present invention (A11) partial nucleotide sequence

According to embodiment 29 described methods, carry out PCR as template with by the use primer to 14 by the chromosomal DNA that will in embodiment 31 (1), prepare.DNA with amplification is connected to cloning vector pCRII-TOPO (Invitrogen Company) and imports intestinal bacteria TOP 10F ' then according to the incidental specification sheets of described carrier.Use Qiagen Tip20 (Qiagen Company) from the intestinal bacteria transformant that obtains, to prepare plasmid DNA.According to the incidental specification sheets of described test kit, use has the primer of nucleotide sequence shown in the SEQ ID NO:57 and has the primer of nucleotide sequence shown in the SEQ ID NO:59, stops cycle sequencing FS easy reaction test kit (AppliedBiosystems Japanese firm) with dyestuff and carries out sequencing reaction.Sequencing reaction uses the plasmid that obtains as template.With dna sequencing instrument 3100 (Applied Biosystems Japanese firm) analytical reaction product.As a result, be provided at represented nucleotide sequence in the Nucleotide 316 to 1048 of nucleotide sequence shown in the SEQ ID NO:139.

In addition, the chromosomal DNA that in embodiment 31 (1), prepares with restriction enzyme PvuII digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 (Universal Genome Walker test kit (Clontech Company)) of nucleotide sequence shown in the SEQ ID NO:161 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, by using a PCR product to have the oligonucleotide and the primer AP2 (UniversalGenome Walker test kit (Clontech Company)) of nucleotide sequence shown in the SEQ ID NO:162, under embodiment 26 (3) described conditions, carry out PCR as template with by use.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1 to 330 of nucleotide sequence shown in the SEQ ID NO:144.

In addition, the chromosomal DNA that in embodiment 31 (1), prepares with restriction enzyme HincII digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 (Universal Genome Walker test kit (Clontech Company)) of nucleotide sequence shown in the SEQ ID NO:163 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, by using a PCR product to have the oligonucleotide and the primer AP2 (UniversalGenome Walker test kit (Clontech Cormpany)) of nucleotide sequence shown in the SEQ IDNO:164, under embodiment 26 (3) described conditions, carry out PCR as template with by use.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 983 to 1449 of nucleotide sequence shown in the SEQ ID NO:144.

(3) sequential analysis of DNA of the present invention (A11)

By the nucleotide sequence that the nucleotide sequence acquisition that provided by embodiment 31 (2) DNA that obtain is represented in SEQ IDNO:144 is provided.There are two open reading-frame (ORF)s (ORF).Therefore, comprise by 1230 Nucleotide (comprising terminator codon) and form and the nucleotide sequence (SEQ ID NO:139) of 409 amino-acid residues (the SEQ IDNO:159) that encode and form and the nucleotide sequence (SEQ ID NO:154) of 68 amino-acid residues of encoding (SEQ ID NO:149) by 207 Nucleotide (comprising terminator codon).The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:139 (SEQ ID NO:159) it is calculated that and is 45177Da.In addition, the proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:154 (SEQ ID NO:149) it is calculated that and is 7147Da.

Embodiment 32 expression of protein of the present invention (A11) in intestinal bacteria

(1) has the generation of the transformed into escherichia coli of protein of the present invention (A11)

By will in embodiment 31 (1), carrying out PCR as template with by use Expand HiFi PCR System (Boehringer ManheimCompany) by the chromosomal DNA from black walnut streptomycete IFO13445 preparation.As primer, use oligonucleotide with nucleotide sequence shown in the SEQ ID NO:165 and pairing with oligonucleotide of nucleotide sequence shown in the SEQ ID NO:166.Reaction soln is formed and maintenance is similar to embodiment 27 (1) described situations.Reaction soln after keeping is carried out 1% agarose gel electrophoresis.Recovery comprises the gel area of the DNA of about 1.5kbp.By use the QIA PhastGel to extract test kit (Qiagen company) purify DNA from the gel that reclaims according to incidental specification sheets.According to the incidental specification sheets of described carrier the DNA that obtains is connected to cloning vector pCRII-TOPO (Invitrogen company) and imports intestinal bacteria TOP10F '.Use Qiagen Tip20 (Qiagen company) from the intestinal bacteria transformant that obtains, to prepare plasmid DNA.According to the incidental specification sheets of described test kit, use and have SEQ ID NO:57 respectively, the oligonucleotide of nucleotide sequence shown in 59 and 186 stops cycle sequencing FS easy reaction test kit (Applied Biosystems Japanese firm) with dyestuff and carries out sequencing reaction as primer.Sequencing reaction uses the plasmid DNA that obtains as template.With dna sequencing instrument 3100 (Applied Biosystems Japanese firm) analytical reaction product.Based on the result, will have the plasmid called after pCR849AF of nucleotide sequence shown in the SEQ ID NO:144.

Next, with restriction enzyme NdeI and HindIII digestion pCR849AF.Digestion product is carried out agarose gel electrophoresis.Cutting comprises the gel area of about 1.5kbp DNA from gel.By use the QIA PhastGel to extract test kit (Qiagen company) purify DNA from the gel that reclaims according to incidental specification sheets.Connect the DNA and the plasmid pKSN2 of the acquisition that digests with NdeI and HindIII and import e. coli jm109 with connecting test kit version 2 (Takara Shuzo Company) according to the incidental specification sheets of described test kit.Prepare plasmid DNA from the intestinal bacteria transformant that obtains.Analyze its structure.Will be wherein between the NdeI site and HindIII site of about 1.5kbp DNA insertion pKSN2 of code book invention protein (A11), comprise the plasmid called after pKSN849AF of nucleotide sequence shown in the SEQ ID NO:144.Plasmid pKSN849AF is imported e. coli jm109.With the intestinal bacteria transformant called after JM109/pKSN849AF that obtains.In addition, plasmid pKSN2 is imported e. coli jm109.The intestinal bacteria transformant called after JM109/pKSN2 that obtains.

(2) expression and the described recovery of protein of protein of the present invention (A11) in intestinal bacteria

Be similar to embodiment 4 (2), cultivate each of e. coli jm109/pKSN849AF and JM109/pKSN2.Reclaim cell.The preparation cell pyrolysis liquid.Under embodiment 4 (2) described methods, preparation supernatant liquor fraction from cell pyrolysis liquid (below, to be called " intestinal bacteria pKSN849AF extract " available from the supernatant liquor fraction of e. coli jm109/pKSN849AF, will be called " intestinal bacteria pKSN2 extract ") available from the supernatant liquor fraction of e. coli jm109/pKSN2.

Prepare the reaction soln of 30 μ l and remain on 30 ℃ following 10 minutes.Reaction soln is made up of 0.1M potassium phosphate buffer (pH7.0), and it comprises 3ppm and uses ¹⁴The compound of C mark (II), 2mM β-NADPH (hereinafter referred to as " component A ") (Oriental Yeast Company), 2mg/ml derives from the ferredoxin (hereinafter referred to as " B component ") (Sigma Company) of spinach, the supernatant liquor fraction that 0.1U/ml ferredoxin reductase (hereinafter referred to as " component C ") (Sigma Company) and 23 μ l reclaim in embodiment 32 (2).Be similar to embodiment 4 (3), the reaction soln after keeping with ethyl acetate extraction also carries out TLC with extract layer and analyzes.After launching the TLC plate, check on it corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).From the reaction soln that comprises intestinal bacteria pKSN849AF extract, detect spot corresponding to compound (III).Contrast does not detect this spot in the reaction soln that comprises intestinal bacteria pKSN2 extract therewith.

Embodiment 33 obtains DNA of the present invention (A12)

(1) preparation of the chromosomal DNA of Tianjin island chain mould IFO 13782

According to embodiment 31 (1) described methods, the chromosomal DNA of preparation Tianjin island chain mould IFO 13782.

(2) has the separation of the DNA of DNA of the present invention (A12) partial nucleotide sequence

According to embodiment 29 described methods, carry out PCR as template with by the use primer to 14 by Tianjin island chain mould IFO 13782 chromosomal DNAs that will in embodiment 33 (1), prepare.Be similar to embodiment 31 (2), with the amplification dna clone to cloning vector pCRII-TOPO (InvitrogenCompany).Analyze its nucleotide sequence.As a result, be provided at represented nucleotide sequence in the Nucleotide 364 to 1096 of nucleotide sequence shown in the SEQ ID NO:140.

In addition, the chromosomal DNA that in embodiment 33 (1), prepares with restriction enzyme SmaI digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:167 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:168 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1 to 392 of nucleotide sequence shown in the SEQ ID NO:145.

In addition, the chromosomal DNA that in embodiment 33 (1), prepares with restriction enzyme PvuII digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:169 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:170 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1048 to 1480 of nucleotide sequence shown in the SEQ ID NO:145.

(3) sequential analysis of DNA of the present invention (A12)

By the nucleotide sequence that the nucleotide sequence acquisition that provided by embodiment 33 (2) DNA that obtain is represented in SEQ IDNO:145 is provided.In described nucleotide sequence, there are two open reading-frame (ORF)s (ORF).Therefore, comprise by 1278 Nucleotide (comprising terminator codon) and form and the nucleotide sequence (SEQ ID NO:140) of 425 amino-acid residues of encoding (SEQ ID NO:160) and form and the nucleotide sequence (SEQ ID NO:155) of 65 amino-acid residues (the SEQ IDNO:150) that encode by 198 Nucleotide (comprising terminator codon).The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:140 (SEQ ID NO:160) it is calculated that and is 46549Da.In addition, the proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:155 (SEQ ID NO:150) it is calculated that and is 6510Da.

Embodiment 34 expression of DNA of the present invention (A12) in intestinal bacteria

(1) has the generation of the transformed into escherichia coli of DNA of the present invention (A12)

Be similar to embodiment 32 (1), except will be in embodiment 33 (1) carrying out PCR as the template and the oligonucleotide that will have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:171 and have a nucleotide sequence shown in the SEQ ID NO:172 as the primer from the chromosomal DNA of Tianjin island chain mould IFO13782 preparation.Be similar to embodiment 32 (1), purify DNA and being cloned among the cloning vector pCRII-TOPO (Invitrogen company) from the PCR reaction soln.With having SEQ IDNO:57 respectively, the nucleotide sequence of the plasmid DNA that the oligonucleotide analysis of nucleotide sequence shown in 59,171,172 and 187 obtains.Based on the result who obtains, will contain the plasmid called after pCR1618F of nucleotide sequence shown in the SEQ ID NO:145.Be similar to embodiment 32 (1), with restriction enzyme NdeI and HindIII digestion pCR1618F.The DNA of the about 1.5kbp of purifying from digestion product.To be connected with plasmid pKSN2 to obtain to comprise the plasmid of nucleotide sequence shown in the SEQ IDNO:145 with the DNA of the NdeI and the acquisition of HindIII digestion, wherein the DNA of code book invention protein (A12) is inserted NdeI site and HindIII site (hereinafter referred to as " pKSN1618F ") of pKSN2.Described plasmid is imported e. coli jm109.The intestinal bacteria transformant that obtains is called JM109/pKSN1618F.

(2) expression and the described recovery of protein of protein of the present invention (A12) in intestinal bacteria

Be similar to embodiment 4 (2), cultivate among e. coli jm109/pKSN1618F and the JM109/pKSN2 each.Reclaim cell.The preparation cell pyrolysis liquid.Under embodiment 4 (2) described methods, preparation supernatant liquor fraction from cell pyrolysis liquid (below, to be called " intestinal bacteria pKSN1618F extract " available from the supernatant liquor fraction of e. coli jm109/pKSN1618F, will be called " intestinal bacteria pKSN2 extract ") available from the supernatant liquor fraction of e. coli jm109/pKSN2.

Prepare the reaction soln of 30 μ l and remain on 30 ℃ following 10 minutes.Except using the supernatant liquor fraction (intestinal bacteria pKSN1618F extract or intestinal bacteria pKSN2 extract) that in embodiment 34 (2), reclaims, be similar to embodiment 32 (3) preparation feedback solution.Analyze with the reaction soln after the ethyl acetate extraction maintenance and with extract layer TLC.After launching the TLC plate, check on it corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).From the reaction soln that comprises intestinal bacteria pKSN1618F extract, detect spot corresponding to compound (III).Contrast does not detect this spot in the reaction soln that comprises intestinal bacteria pKSN2 extract therewith.

Embodiment 35 obtains DNA of the present invention (A13)

(1) preparation of the chromosomal DNA of hot lightskyblue streptomycete IFO 14273t

According to embodiment 31 (1) described methods, prepare the chromosomal DNA of hot lightskyblue streptomycete IFO 14273t.

(2) has the separation of the DNA of DNA of the present invention (A13) partial nucleotide sequence

According to embodiment 29 described methods, carry out PCR as template with by the use primer to 14 by the hot lightskyblue streptomycete IFO 14273t chromosomal DNA that will in embodiment 35 (1), prepare.Be similar to embodiment 31 (2), with the amplification dna clone to cloning vector pCRII-TOPO (Invitrogen Company).Analyze its nucleotide sequence.As a result, be provided at represented nucleotide sequence in the Nucleotide 295 to 1027 of nucleotide sequence shown in the SEQ ID NO:141.

In addition, the chromosomal DNA that in embodiment 35 (1), prepares with restriction enzyme HincII digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:173 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:174 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1 to 370 of nucleotide sequence shown in the SEQ ID NO:146.

In addition, the chromosomal DNA that in embodiment 35 (1), prepares with restriction enzyme SmaI digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:175 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:176 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 960 to 1473 of nucleotide sequence shown in the SEQ ID NO:146.

(3) sequential analysis of DNA of the present invention (A13)

By the nucleotide sequence that the nucleotide sequence acquisition that provided by embodiment 35 (2) DNA that obtain is represented in SEQ IDNO:146 is provided.In described nucleotide sequence, there are two open reading-frame (ORF)s (ORF).Therefore, comprise by 1209 Nucleotide (comprising terminator codon) and form and the nucleotide sequence (SEQ ID NO:141) of 402 amino-acid residues of encoding (SEQ ID NO:136) and form and the nucleotide sequence (SEQ ID NO:156) of 83 amino-acid residues (the SEQ IDNO:151) that encode by 252 Nucleotide (comprising terminator codon).The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQIDNO:141 (SEQID NO:136) it is calculated that and is 44629Da.In addition, the proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:156 (SEQ ID NO:151) it is calculated that and is 8635Da.

Embodiment 36 expression of protein of the present invention (A13) in intestinal bacteria

(1) has the generation of the transformed into escherichia coli of protein of the present invention (A13)

Be similar to embodiment 32 (1), except will be in embodiment 35 (1) carrying out PCR as the template and the oligonucleotide that will have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:177 and have a nucleotide sequence shown in the SEQ ID NO:178 as the primer from the chromosomal DNA of hot lightskyblue streptomycete IFO 14273t preparation.Be similar to embodiment 32 (1), purify DNA and being cloned among the cloning vector pCRII-TOPO (Invitrogen company) from the PCR reaction soln.With having SEQ ID NO:57 respectively, the nucleotide sequence of the plasmid DNA that the oligonucleotide analysis of nucleotide sequence shown in 59,173,175 and 188 obtains.Based on the result who obtains, will have the plasmid called after pCR474F of nucleotide sequence shown in the SEQ ID NO:146.Be similar to embodiment 32 (1), with restriction enzyme NdeI and HindIII digestion pCR474F.The DNA of the about 1.5kbp of purifying from digestion product.To be connected with plasmid pKSN2 to obtain to comprise the plasmid of nucleotide sequence shown in the SEQ ID NO:146 with the DNA of the NdeI and the acquisition of HindIII digestion, wherein the DNA of code book invention protein (A13) is inserted NdeI site and HindIII site (hereinafter referred to as " pKSN474F ") of pKSN2.Described plasmid is imported e. coli jm109.The intestinal bacteria transformant that obtains is called JM109/pKSN474F.

(2) expression and the described recovery of protein of protein of the present invention (A13) in intestinal bacteria

Be similar to embodiment 4 (2), cultivate among e. coli jm109/pKSN474F and the JM109/pKSN2 each.Reclaim cell.The preparation cell pyrolysis liquid.Under embodiment 4 (2) described methods, preparation supernatant liquor fraction from cell pyrolysis liquid (below, to be called " intestinal bacteria pKSN474F extract " available from the supernatant liquor fraction of e. coli jm109/pKSN474F, will be called " intestinal bacteria pKSN2 extract ") available from the supernatant liquor fraction of e. coli jm109/pKSN2.

Prepare the reaction soln of 30 μ l and remain on 30 ℃ following 10 minutes.Except using the supernatant liquor fraction (intestinal bacteria pKSN474F extract or intestinal bacteria pKSN2 extract) that in embodiment 36 (2), reclaims, be similar to embodiment 32 (3) preparation feedback solution.Analyze with the reaction soln after the ethyl acetate extraction maintenance and with extract layer TLC.After launching the TLC plate, check on it corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).From the reaction soln that comprises intestinal bacteria pKSN474F extract, detect spot corresponding to compound (III).Contrast does not detect this spot in the reaction soln that comprises intestinal bacteria pKSN2 extract therewith.

Embodiment 37 obtains DNA of the present invention (A14)

According to embodiment 29 described methods, produce look streptomycete IFO 13673t chromosomal DNA as template with by using primer to carry out PCR to 14 by the pelletizing that will in embodiment 37 (1), prepare.Be similar to embodiment 31 (2), with the amplification dna clone to cloning vector pCRII-TOPO (Invitrogen Company).Analyze its nucleotide sequence.As a result, be provided at represented nucleotide sequence in the Nucleotide 316 to 1048 of nucleotide sequence shown in the SEQ ID NO:142.

In addition, the chromosomal DNA that in embodiment 37 (1), prepares with restriction enzyme SmaI digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:179 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:180 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1 to 330 of nucleotide sequence shown in the SEQ ID NO:147.

In addition, the chromosomal DNA that in embodiment 37 (1), prepares with restriction enzyme HincII digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:181 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:182 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 982 to 1449 of nucleotide sequence shown in the SEQ ID NO:147.

(3) sequential analysis of DNA of the present invention (A14)

By the nucleotide sequence that the nucleotide sequence acquisition that provided by embodiment 37 (2) DNA that obtain is represented in SEQ IDNO:147 is provided.In described nucleotide sequence, there are two open reading-frame (ORF)s (ORF).Therefore, comprise by 1230 Nucleotide (comprising terminator codon) and form and the nucleotide sequence (SEQ ID NO:142) of 409 amino-acid residues of encoding (SEQ ID NO:137) and form and the nucleotide sequence (SEQ ID NO:157) of 68 amino-acid residues (the SEQ IDNO:152) that encode by 207 Nucleotide (comprising terminator codon).The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:142 (SEQ ID NO:137) it is calculated that and is 45089Da.In addition, the proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:157 (SEQ ID NO:152) it is calculated that and is 7174Da.

Embodiment 38 expression of DNA of the present invention (A14) in intestinal bacteria

(1) has the generation of the transformed into escherichia coli of DNA of the present invention (A14)

Be similar to embodiment 32 (1), carry out PCR as the template and the oligonucleotide that will have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:183 and have a nucleotide sequence shown in the SEQ ID NO:184 as the primer in embodiment 37 (1) except the pelletizing that will prepare produces look streptomycete IFO13673t chromosomal DNA.Be similar to embodiment 32 (1), purify DNA and being cloned among the cloning vector pCRII-TOPO (Invitrogen company) from the PCR reaction soln.With having SEQ IDNO:57 respectively, the nucleotide sequence of the plasmid DNA that the oligonucleotide analysis of nucleotide sequence shown in 59 and 189 obtains.Based on the result who obtains, will have the plasmid called after pCR1491AF of nucleotide sequence shown in the SEQ ID NO:147.Be similar to embodiment 32 (1), with restriction enzyme NdeI and HindIII digestion pCR1491AF.The DNA of the about 1.5kbp of purifying from digestion product.To be connected with plasmid pKSN2 to obtain to comprise the plasmid of nucleotide sequence shown in the SEQ ID NO:147 with the DNA of the NdeI and the acquisition of HindIII digestion, wherein the DNA of code book invention protein (A14) is inserted NdeI site and HindIII site (hereinafter referred to as " pKSN1491AF ") of pKSN2.Described plasmid is imported e. coli jm109.The intestinal bacteria transformant that obtains is called JM109/pKSN1491AF.

(2) expression and the described recovery of protein of protein of the present invention (A14) in intestinal bacteria

Be similar to embodiment 4 (2), cultivate among e. coli jm109/pKSN1491AF and the JM109/pKSN2 each.Reclaim cell.The preparation cell pyrolysis liquid.Under embodiment 4 (2) described methods, preparation supernatant liquor fraction from cell pyrolysis liquid (below, to be called " intestinal bacteria pKSN1491AF extract " available from the supernatant liquor fraction of e. coli jm109/pKSN1491AF, will be called " intestinal bacteria pKSN2 extract ") available from the supernatant liquor fraction of e. coli jm109/pKSN2.

Prepare the reaction soln of 30 μ l and remain on 30 ℃ following 10 minutes.Except using the supernatant liquor fraction (intestinal bacteria pKSN1491AF extract or intestinal bacteria pKSN2 extract) that in embodiment 38 (2), reclaims, be similar to embodiment 32 (3) preparation feedback solution.Analyze with the reaction soln after the ethyl acetate extraction maintenance and with extract layer TLC.After launching the TLC plate, check on it corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).From the reaction soln that comprises intestinal bacteria pKSN1491AF extract, detect spot corresponding to compound (III).Contrast does not detect this spot in the reaction soln that comprises intestinal bacteria pKSN2 extract therewith.

Embodiment 39 obtains DNA of the present invention (A15)

(1) olive produces the preparation of the chromosomal DNA of look streptomycete IFO12444

According to embodiment 31 (1) described methods, the preparation olive produces the chromosomal DNA of look streptomycete IFO 12444.

(2) has the separation of the DNA of DNA of the present invention (A15) partial nucleotide sequence

According to embodiment 29 described methods, produce look streptomycete IFO 12444 chromosomal DNAs template with by using primer to carry out PCR to 14 by the olive that will in embodiment 39 (1), prepare.Be similar to embodiment 31 (2), with the amplification dna clone to cloning vector pCRII-TOPO (Invitrogen Company).Analyze its nucleotide sequence.As a result, be provided at represented nucleotide sequence in the Nucleotide 316 to 1048 of nucleotide sequence shown in the SEQ ID NO:143.

In addition, the chromosomal DNA that in embodiment 37 (1), prepares with restriction enzyme SmaI digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:179 by the DNA that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:180 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1 to 330 of nucleotide sequence shown in the SEQ ID NO:148.

In addition, the chromosomal DNA that in embodiment 39 (1), prepares with restriction enzyme SmaI digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:181 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:182 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 982 to 1449 of nucleotide sequence shown in the SEQ ID NO:148.

(3) sequential analysis of DNA of the present invention (A15)

By the nucleotide sequence that the nucleotide sequence acquisition that provided by embodiment 39 (2) DNA that obtain is represented in SEQ IDNO:148 is provided.In described nucleotide sequence, there are two open reading-frame (ORF)s (ORF).Therefore, comprise by 1230 Nucleotide (comprising terminator codon) and form and the nucleotide sequence (SEQ ID NO:143) of 409 amino-acid residues of encoding (SEQ ID NO:138) and form and the nucleotide sequence (SEQ ID NO:158) of 68 amino-acid residues (the SEQ IDNO:153) that encode by 207 Nucleotide (comprising terminator codon).The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:143 (SEQ ID NO:138) it is calculated that and is 45116Da.In addition, the proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:158 (SEQ ID NO:153) it is calculated that and is 7179Da.

Embodiment 40 expression of DNA of the present invention (A15) in intestinal bacteria

(1) has the generation of the transformed into escherichia coli of DNA of the present invention (A15)

Be similar to embodiment 32 (1), carry out PCR as the template and the oligonucleotide that will have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:184 and have a nucleotide sequence shown in the SEQ ID NO:185 as the primer in embodiment 39 (1) except the olive that will prepare produces look streptomycete IFO12444 chromosomal DNA.Be similar to embodiment 32 (1), purify DNA and being cloned among the cloning vector pCRJI-TOPO (Invitrogen company) from the PCR reaction soln.With having SEQ IDNO:57 respectively, the nucleotide sequence of the plasmid DNA that the oligonucleotide analysis of nucleotide sequence shown in 59 and 189 obtains.Based on the result who obtains, will have the plasmid called after pCR1555AF of nucleotide sequence shown in the SEQ ID NO:148.Be similar to embodiment 32 (1), with restriction enzyme NdeI and HindIII digestion pCR1555AF.The DNA of the about 1.5kbp of purifying from digestion product.To be connected with plasmid pKSN2 to obtain to comprise the plasmid of nucleotide sequence shown in the SEQ ID NO:148 with the DNA of the NdeI and the acquisition of HindIII digestion, wherein the DNA of code book invention protein (A15) is inserted NdeI site and HindIII site (hereinafter referred to as " pKSN1555AF ") of pKSN2.Described plasmid is imported e. coli jm109.The intestinal bacteria transformant that obtains is called JM109/pKSN1555AF.

(2) expression and the described recovery of protein of protein of the present invention (A15) in intestinal bacteria

Be similar to embodiment 4 (2), cultivate among e. coli jm109/pKSN1555AF and the JM109/pKSN2 each.Reclaim cell.The preparation cell pyrolysis liquid.Under embodiment 4 (2) described methods, preparation supernatant liquor fraction from cell pyrolysis liquid (below, to be called " intestinal bacteria pKSN1555AF extract " available from the supernatant liquor fraction of e. coli jm109/pKSN1555AF, will be called " intestinal bacteria pKSN2 extract ") available from the supernatant liquor fraction of e. coli jm109/pKSN2.

Prepare the reaction soln of 30 μ 1 and remain on 30 ℃ following 10 minutes.Except using the supernatant liquor fraction (intestinal bacteria pKSN1555AF extract or intestinal bacteria pKSN2 extract) that in embodiment 40 (2), reclaims, be similar to embodiment 32 (3) preparation feedback solution.Analyze with the reaction soln after the ethyl acetate extraction maintenance and with extract layer TLC.After launching the TLC plate, check on it corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (IH) (Rf value 0.24 and 0.29).From the reaction soln that comprises intestinal bacteria pKSN1555AF extract, detect spot corresponding to compound (III).Contrast does not detect this spot in the reaction soln that comprises intestinal bacteria pKSN2 extract therewith.

Embodiment 41 is by the metabolism of the compound of protein of the present invention (A1)

(1) preparation of plastid fraction

One hectogram (100g) radish greenery seeds (Takii Seed) are cut in the laboratory sheets rag moist in the pallet, in 25 ℃ of following dark, cultivated 6 days, under luminescent lamp, cultivated 4 hours then.With Nissei AM-8 homogenizer (Nihonseiki Seisakusho; 18,000-20,000rpm, 4 ℃, 5 seconds) at the damping fluid that breaks (1mM magnesium chloride, 20mM N-three (methylol) methyl-2-aminoethane sulfonic acid ester, 10mM N-2-hydroxyethyl piperidine-N '-2-ethylsulfonic acid ester, 0.5mM EDTA, 5mM halfcystine, 0.5M sucrose; PH7.7) grind 30 grams (30g) in and newly turn green cotyledon.The cell pyrolysis liquid that obtains is passed through 4 layers of nylon gause.With the solution centrifugal (13,170xg, 1 minute) that obtains.With 60ml resistates fraction and centrifugal (2,640xg, 4 ℃, 2 minutes) that damping fluid suspend to obtain of breaking.The resistates fraction is resuspended in 10ml breaks in the damping fluid, in centrifuge tube, use high-density damping fluid (1mM magnesium chloride, 20mMN-three (methylol) methyl-2-aminoethane sulfonic acid ester, 30mM N-2-hydroxyethyl piperidine-N '-2-ethylsulfonic acid ester, 0.5mM EDTA, the 5mM halfcystine, 0.6M sucrose; PH7.7) layering, and centrifugal (675xg, 4 ℃, 15 minutes).Resistates is suspended in 3ml buffer suspension liquid (1mM magnesium chloride, 20mM N-three (methylol) methyl-2-aminoethane sulfonic acid ester, 30mM N-2-hydroxyethyl piperidine-N '-2-ethylsulfonic acid ester, 0.5mM EDTA; PH7.7) in and be called the plastid fraction.

(2) metabolism of the compound (XII) by protein of the present invention (A1)

100 μ l reaction solns of preparation 50mM potassiumphosphate (pH 7.0), it comprises 5ppm compound (XII), 3mM β-NADPH (hereinafter referred to as " component A ") (Oriental Yeast Company), the ferredoxin (hereinafter referred to as " B component ") that 1mg/ml derives from spinach (SigmaCompany), 0.15U/ml ferredoxin reductase (hereinafter referred to as " component C ") is (SigmaCompany) and the supernatant liquor fraction that reclaims in embodiment 4 (2) of 20 μ l.With reaction soln remain on 30 ℃ following 10 minutes.In addition, prepare similarly and keep not to add be used for above-mentioned reaction soln composition, be selected from component A, 100 μ l reaction solns of the 50mM potassium phosphate buffer (pH 7.0) of at least a component of B component and component C and the supernatant liquor fraction that among embodiment 4 (2), prepares.Ten microlitres (10 μ l) 2N HCl and 500 μ l ethyl acetate are added and be mixed in the every kind of reaction soln that keeps later.8, under the 000xg that the reaction soln that produces is centrifugal to reclaim 490 μ l ethyl acetate layers.After the drying under reduced pressure ethyl acetate layer, resistates is dissolved in the potassium phosphate buffer (pH7.0) of 100 μ l50mM.Analyze on the HPLC 40 microlitres (40 μ l) fraction solution (below, self-contained component A in the future, B component, the fraction solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 4 (2) is called " (XII) metabolism solution (A1) "; In addition, will not comprise B component from not comprising component A, the fraction solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 4 (2) is called " (XII) contrast solution (A1) ").Compare with the concentration of the compound (XII) that detects from (XII) contrast solution (A1), the concentration of the compound (XII) that detects from (XII) metabolism solution (A1) is lower.Detect the peak from (XII) metabolism solution (A1) in addition, it does not detect from (XII) contrast solution (A1).Carry out mass spectroscopy for the compound that in this peak, comprises.The quality that is included in the compound in this peak is littler by 14 than the quality of compound (XII).

Mix above-mentioned (XII) the metabolism solution (A1) of 32 times of 20 microlitres (20 μ l) dilutions and the plastid fraction that 60 μ l prepare in embodiment 41 (1).Under dark condition, add 20 μ l substrate solutions (10mM adenosine triphosphate, 5mM amino-laevulic acid, 4mM glutathione reductase and 0.6mM NAD ⁺PH 6.5; Below, this substrate solution is called " PPO substrate solution ") and under 30 ℃, kept 1.5 hours.In addition, replace (XII) metabolism solution (A1) of 32 times of described 20 μ l dilutions, preparation adds the reaction soln of (XII) contrast solution (A1) of 32 times of 20 μ l dilutions, adds and keep the PPO substrate solution similarly.With 300 (300 μ l) methyl-sulphoxide-carbinol mixture (methyl-sulphoxide: methyl alcohol=7:3) add in each reaction soln that keeps later and centrifugal (8000xg, 4 ℃, 10 minutes).Reclaim supernatant liquor and under following analysis condition, carry out anti-phase HPC analysis to measure the amount of PPIX.The amount of PPIX is more than the amount of PPIX in the reaction soln that adds (XII) contrast solution (A1) in the reaction soln of adding (XII) metabolism solution (A1).

(HPLC analysis condition 2)

Post: SUMIPAX ODS212 (Sumika Chemical Analysis Service)

Flow velocity: 2ml/ minute

Detect wavelength: fluorescence Ex:410nm Em:630nm

Eluent: 95: 5 mixtures (pH5.7) of methyl alcohol and 1M ammonium acetate

(3) metabolism of the compound (XIII) by the present invention (A1)

Except using 5ppm compound (XIII) rather than 5ppm compound (XII), similar embodiment 41 (2) described method preparation and maintenance reaction solns.Be similar to embodiment 41 (2), each in the reaction soln after keeping with ethyl acetate extraction is dissolved in the resistates that obtains in the 100 μ l methyl-sulphoxides.Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 4 (2) is called " (XIII) metabolism solution (A1) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 4 (2) is called " (XIII) contrast solution (A1) ").Compare with the concentration of the compound (XIII) that detects from (XIII) contrast solution (A1), the concentration of the compound (XIII) that detects from (XIII) metabolism solution (A1) is lower.Detect the peak from (XIII) metabolism solution (A1) in addition, it does not detect from (XIII) contrast solution (A1).Carry out mass spectroscopy for the compound that in this peak, comprises.The quality that is included in the compound in this peak is littler by 14 than the quality of compound (XIII).

Above-mentioned (XIII) the metabolism solution (A1) and the 60 μ l plastid fractions of mixing 128 times of 20 microlitres (20 μ l) dilutions.Under dark condition, add 20 μ l PPO substrate solutions and descend maintenance 1.5 hours at 30 ℃.In addition, replace (XIII) metabolism solution (A1) of 128 times of described 20 μ l dilutions, preparation adds the reaction soln of (XIII) contrast solution (A1) of 128 times of 20 μ l dilutions, adds and keep the PPO substrate solution similarly.Be similar to embodiment 4 (2), the various reaction solns after preparation keeps also carry out the reversed-phase HPLC analysis for 2 times to measure the amount of PPIX at above-mentioned analysis condition.The amount of PPIX is more than the amount of PPIX in the reaction soln that adds (XIII) contrast solution (A1) in the reaction soln of adding (XIII) metabolism solution (A1).

(4) metabolism of the compound (XVI) by the present invention (A1)

Except using 12.5ppm compound (XVI) rather than 5ppm compound (XII), similar embodiment 41 (2) described method preparation and maintenance reaction solns.Be similar to embodiment 41 (2), each in the reaction soln after keeping with ethyl acetate extraction is dissolved in the resistates that obtains in the 200 μ l50mM potassium phosphate buffers (pH 7.0).Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 4 (2) is called " (XVI) metabolism solution (A1) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 4 (2) is called " (XVI) contrast solution (A1) ").Compare with the concentration of the compound (XVI) that detects from (XVI) contrast solution (A1), the concentration of the compound (XVI) that detects from (XVI) metabolism solution (A1) is lower.Detect the peak from (XVI) metabolism solution (A1) in addition, it does not detect from (XVI) contrast solution (A1).

Above-mentioned (XVI) the metabolism solution (A1) and the 60 μ l plastid fractions of mixing 8 times of 20 microlitres (20 μ l) dilutions.Under dark condition, add 20 μ l PPO substrate solutions and descend maintenance 1.5 hours at 30 ℃.In addition, replace (XVI) metabolism solution (A1) of 8 times of described 20 μ l dilutions, preparation adds the reaction soln of (XVI) contrast solution (A1) of 8 times of 20 μ l dilutions, adds and keep the PPO substrate solution similarly.Be similar to embodiment 41 (2), the various reaction solns after preparation keeps also carry out the reversed-phase HPLC analysis for 2 times to measure the amount of PPIX at above-mentioned analysis condition.The amount of PPIX is more than the amount of PPIX in the reaction soln that adds (XVI) contrast solution (A1) in the reaction soln of adding (XVI) metabolism solution (A1).

(5) metabolism of the compound (XVII) by protein of the present invention (A1)

Except using 12.5ppm compound (XVII) rather than 5ppm compound (XII), similar embodiment 41 (2) described method preparation and maintenance reaction solns.Be similar to embodiment 41 (2), each in the reaction soln after keeping with ethyl acetate extraction is dissolved in the resistates that obtains in the 200 μ l50mM potassium phosphate buffers (pH 7.0).Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 4 (2) is called " (XVII) metabolism solution (A1) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 4 (2) is called " (XVII) contrast solution (A1) ").Compare with the concentration of the compound (XVII) that detects from (XVII) contrast solution (A1), the concentration of the compound (XVII) that detects from (XVII) metabolism solution (A1) is lower.Detect the peak from (XVII) metabolism solution (A1) in addition, it does not detect from (XVII) contrast solution (A1).

Above-mentioned (XVII) the metabolism solution (A1) and the 60 μ l plastid fractions of mixing 32 times of 20 microlitres (20 μ l) dilutions.Under dark condition, add 20 μ l PPO substrate solutions and descend maintenance 1.5 hours at 30 ℃.In addition, replace (XVII) metabolism solution (A1) of 32 times of described 20 μ l dilutions, preparation adds the reaction soln of (XVII) contrast solution (A1) of 32 times of 20 μ l dilutions, adds and keep the PPO substrate solution similarly.Be similar to embodiment 41 (2), the various reaction solns after preparation keeps also carry out the reversed-phase HPLC analysis for 2 times to measure the amount of PPIX at above-mentioned analysis condition.The amount of PPIX is more than the amount of PPIX in the reaction soln that adds (XVII) contrast solution (A1) in the reaction soln of adding (XVII) metabolism solution (A1).

(6) metabolism of the compound (VI) by protein of the present invention (A1)

E. coli jm109/pKSN657F is being comprised overnight incubation in the TB substratum of 50 μ g/ml penbritins at 3ml under 37 ℃.The media transfer that one milliliter (1ml) obtained comprises to 100ml in the TB substratum of 50 μ g/ml penbritins and at 26 ℃ cultivates down.Work as OD ₆₆₀Reach at about 0.5 o'clock, the 5-amino-laevulic acid is added to the final concentration of 500 μ M, continue to cultivate.After 30 (30) minutes, add the final concentration of IPTG to 1mM, further cultivated 20 hours.

From substratum, reclaim cell, wash and be suspended among the 0.1M Tris-HCl that 10ml comprises 1% glucose with 0.1M tris-HCl damping fluid (pH 7.5).The cell suspension that compound (VI) is added acquisition is to the final concentration of 100ppm and at 30 ℃ of incubations that vibrate down.Respectively when starting of oscillation 0 hour and 1 day, fractional separation 2ml cell suspension.Each adds 50 microlitres (50 μ l) 2N HCl and uses the 2ml ethyl acetate extraction.On HPLC, analyze the ethyl acetate layer that obtains 1 time at reaction conditions.The concentration of the compound (VI) that detects with 0 hour the ethyl acetate layer of cell suspension preparation behind starting of oscillation is compared, and the concentration of the compound (VI) that the ethyl acetate layer of 1 day cell suspension preparation detects behind the starting of oscillation is lower.In addition, the ethyl acetate layer of 1 day cell suspension preparation detects the peak behind the starting of oscillation, and it does not detect in 0 hour the ethyl acetate layer of cell suspension preparation behind starting of oscillation.Be included in the mass spectroscopy of the compound in the described peak.The quality that is included in the compound in the described peak is littler by 14 than the quality of compound (VI).

(7) metabolism of the compound (VIII) by protein of the present invention (A1)

Except using compound (VIII) rather than compound (VI), carry out the cultivation of e. coli jm109/pKSN657F according to embodiment 41 (6) described methods, the preparation of cell suspension, the vibration incubation that adds the cell suspension of compound (VIII) is analyzed from the reagent preparation of cell suspension and the HPLC of reagent.The concentration of the compound (VIII) that detects with 0 hour the ethyl acetate layer of cell suspension preparation behind starting of oscillation is compared, and the concentration of the compound (VIII) that the ethyl acetate layer of 1 day cell suspension preparation detects behind the starting of oscillation is lower.In addition, the ethyl acetate layer of 1 day cell suspension preparation detects two peaks behind the starting of oscillation, and it does not detect in 0 hour the ethyl acetate layer of cell suspension preparation behind starting of oscillation.Be included in the mass spectroscopy of the compound in the described peak.The quality of the compound that one of is included in the described peak is littler by 14 than the quality of compound (VIII), and the quality that is included in the compound in another peak is littler by 28 than the quality of compound (VIII).

(8) metabolism of the compound (X) by protein of the present invention (A1)

Except using compound (X) rather than compound (VI), carry out the cultivation of e. coli jm109/pKSN657F according to embodiment 41 (6) described methods, the preparation of cell suspension, the shaking culture that adds the cell suspension of compound (X) is analyzed from the reagent preparation of cell suspension and the HPLC of reagent.The concentration of the compound (X) that detects with 0 hour the ethyl acetate layer of cell suspension preparation behind starting of oscillation is compared, and the concentration of the compound (X) that the ethyl acetate layer of 1 day cell suspension preparation detects behind the starting of oscillation is lower.In addition, the ethyl acetate layer of 1 day cell suspension preparation detects two peaks behind the starting of oscillation, and it does not detect in 0 hour the ethyl acetate layer of cell suspension preparation behind starting of oscillation.Be included in the mass spectroscopy of the compound in the described peak.The quality of the compound that one of is included in the described peak is littler by 40 than the quality of compound (X), and the quality that is included in the compound in another peak is littler by 54 than the quality of compound (X).

(9) metabolism of the compound (XI) by protein of the present invention (A1)

Except using compound (XI) rather than compound (VI), carry out the cultivation of e. coli jm109/pKSN657F according to embodiment 41 (6) described methods, the preparation of cell suspension, the shaking culture that adds the cell suspension of compound (XI) is analyzed from the reagent preparation of cell suspension and the HPLC of reagent.The concentration of the compound (XI) that detects with 0 hour the ethyl acetate layer of cell suspension preparation behind starting of oscillation is compared, and the concentration of the compound (XI) that the ethyl acetate layer of 1 day cell suspension preparation detects behind the starting of oscillation is lower.In addition, the ethyl acetate layer of 1 day cell suspension preparation detects two peaks behind the starting of oscillation, and it does not detect in 0 hour the ethyl acetate layer of cell suspension preparation behind starting of oscillation.Be included in the mass spectroscopy of the compound in the described peak.The quality of the compound that one of is included in the described peak is littler by 14 than the quality of compound (XI), and the quality that is included in the compound in another peak is littler by 16 than the quality of compound (XI).

Embodiment 42 is by the metabolism of the compound of protein of the present invention (A11)

(1) metabolism of the compound (X) by The compounds of this invention (A11)

To comprise overnight incubation in the TB substratum of 50 μ g/ml penbritins at 3ml under each comfortable 37 ℃ of e. coli jm109/pKSN849AF and the e. coli jm109/pKSN2.The media transfer that one milliliter (1ml) obtained comprises to 100ml in the TB substratum of 50 μ g/ml penbritins and at 26 ℃ cultivates down.Work as OD ₆₆₀Reach at about 0.5 o'clock, the 5-amino-laevulic acid is added to the final concentration of 500 μ M, continue to cultivate.After 30 (30) minutes, add the final concentration of IPTG to 1mM, further cultivated 18 hours.

From substratum, reclaim cell, wash and be suspended among the 0.1M Tris-HCl that 10ml comprises 1% glucose with 0.1M tris-HCl damping fluid (pH7.5).The cell suspension that compound (X) is added acquisition is to the final concentration of 25ppm and at 30 ℃ of incubations that vibrate down.Respectively when starting of oscillation 0 hour and 4 days, fractional separation 2ml cell suspension.Each adds 50 microlitres (50 μ l) 2N HCl and uses the 2ml ethyl acetate extraction.On HPLC, analyze the ethyl acetate layer that obtains 1 time at reaction conditions.The concentration of the compound (X) that detects with ethyl acetate layer from JM109/pKSN2 cell suspension preparation is compared, and the concentration of the compound (X) that detects from the ethyl acetate layer of JM109/pKSN849AF cell suspension preparation is lower.In addition, the ethyl acetate layer for preparing from the JM109/pKSN849AF cell suspension detects 3 peaks, and it is not detecting from the ethyl acetate layer of JM109/pKSN2 cell suspension preparation.In 3 peaks, the elution time of peak 1 in HPLC matches with the peak elution time of quality than the compound of the compound (X) little 40 that detects among the embodiment 41 (8).In addition, the HPLC elution time at another peak matches with the peak elution time of quality than the compound of the compound (X) little 54 that detects among the embodiment 41 (8).

After drying, respectively, the ethyl acetate layer that will prepare from above-mentioned JM109/pKSN849AF cell suspension from the 1ml ethyl acetate layer and the 1ml of above-mentioned JM109/pKSN2 cell suspension preparation, resistates be dissolved in the 1ml methyl-sulphoxide (below, the solution since the ethyl acetate layer of JM109/pKSN849AF preparation is called " (X) metabolism solution (A11) " in the future; In addition, in the future be called " (X) contrast solution (A11) " since the solution of the ethyl acetate layer of JM109/pKSN2 cell suspension preparation).

Above-mentioned (X) the metabolism solution (A11) and the 60 μ l plastid fractions of mixing 128 times of 20 microlitres (20 μ l) dilutions.Under dark condition, add 20 μ l PPO substrate solutions and descend maintenance 1.5 hours at 30 ℃.In addition, replace (X) metabolism solution (A11) of 128 times of described 20 μ l dilutions, preparation adds the reaction soln of (X) contrast solution (A11) of 128 times of 20 μ l dilutions, adds and keep the PPO substrate solution similarly.Be similar to embodiment 41 (2), the various reaction solns after preparation keeps also carry out the reversed-phase HPLC analysis for 2 times to measure the amount of PPIX at above-mentioned analysis condition.The amount of PPIX is more than the amount of PPIX in the reaction soln that adds (X) contrast solution (A11) in the reaction soln of adding (X) metabolism solution (A11).

(2) metabolism of the compound (XII) by protein of the present invention (A11)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 4 (2) in embodiment 32 (2), according to embodiment 41 (2) described methods preparations with keep reaction solns.Be similar to embodiment 41 (2), the various reaction solns after keeping with ethyl acetate extraction, and the resistates that obtains is dissolved in the 100 μ l50mM potassium phosphate buffers (pH7.0).Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 32 (2) is called " (XII) metabolism solution (A11) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 32 (2) is called " (XII) contrast solution (A11) ").Compare with the concentration of the compound (XII) that detects from (XII) contrast solution (A11), the concentration of the compound (XII) that detects from (XII) metabolism solution (A11) is lower.Detect the peak from (XII) metabolism solution (A11) in addition, it does not detect from (XII) contrast solution (A11).The peak elution time of the compound of the described compound (XII) little 14 that (XII) detects in the metabolism solution (A1) at embodiment 41 (2) at the elution time at the above peak of HPLC and mass ratio matches.

(3) metabolism of the compound (XIII) by protein of the present invention (A11)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 4 (2) in embodiment 32 (2), according to embodiment 41 (3) described methods preparations with keep reaction solns.Be similar to embodiment 41 (2), the various reaction solns after keeping with ethyl acetate extraction, and the resistates that obtains is dissolved in the 100 μ l methyl-sulphoxides.Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 32 (2) is called " (XIII) metabolism solution (A11) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 32 (2) is called " (XIII) contrast solution (A11) ").Compare with the concentration of the compound (XIII) that detects from (XIII) contrast solution (A11), the concentration of the compound (XIII) that detects from (XIII) metabolism solution (A11) is lower.Detect the peak from (XIII) metabolism solution (A11) in addition, it does not detect from (XIII) contrast solution (A11).The peak elution time of the compound of the described compound (XIII) little 14 that (XIII) detects in the metabolism solution (A11) at embodiment 41 (3) at the elution time at the above peak of HPLC and mass ratio matches.

(4) metabolism of the compound (XVI) by protein of the present invention (A11)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 4 (2) in embodiment 32 (2), according to embodiment 41 (4) described methods preparations with keep reaction solns.Be similar to embodiment 41 (2), the various reaction solns after keeping with ethyl acetate extraction, and the resistates that obtains is dissolved in the 200 μ l 50mM potassium phosphate buffers (pH7.0).Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 32 (2) is called " (XVI) metabolism solution (A11) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 32 (2) is called " (XVI) contrast solution (A11) ").Compare with the concentration of the compound (XVI) that detects from (XVI) contrast solution (A11), the concentration of the compound (XVI) that detects from (XVI) metabolism solution (A11) is lower.Detect the peak from (XVI) metabolism solution (A11) in addition, it does not detect from (XVI) contrast solution (A11).The elution time at the above peak of HPLC with in embodiment 41 (4), from (XVI) metabolism solution (A11), detect and in (XVI) contrast solution (A11) elution time at undetected peak match.

(5) metabolism of the compound (XVII) by protein of the present invention (A11)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 4 (2) in embodiment 32 (2), according to embodiment 41 (5) described methods preparations with keep reaction solns.Be similar to embodiment 41 (2), the various reaction solns after keeping with ethyl acetate extraction, and the resistates that obtains is dissolved in the 200 μ l 50mM potassium phosphate buffers (pH7.0).Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 32 (2) is called " (XVII) metabolism solution (A11) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 32 (2) is called " (XVII) contrast solution (A11) ").Compare with the concentration of the compound (XVII) that detects from (XVII) contrast solution (A11), the concentration of the compound (XVII) that detects from (XVII) metabolism solution (A11) is lower.Detect the peak from (XVII) metabolism solution (A11) in addition, it does not detect from (XVII) contrast solution (A11).The elution time at the above peak of HPLC with in embodiment 41 (5), from (XVII) metabolism solution (A1), detect and in (XVII) contrast solution (A1) elution time at undetected peak match.

Embodiment 43 is by protein of the present invention (A2), (A3), and (A12), (A13), (A14) or (A15) or the metabolism of the compound of protein of the present invention (A10)

(1) metabolism of the compound (XII) by protein of the present invention (A2)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 4 (2) in embodiment 7 (2), according to embodiment 41 (2) described methods preparations with keep reaction solns.Be similar to embodiment 41 (2), the various reaction solns after keeping with ethyl acetate extraction, and the resistates that obtains is dissolved in the 100 μ l50mM potassium phosphate buffers (pH7.0).Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 7 (2) is called " (XII) metabolism solution (A2) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 7 (2) is called " (XII) contrast solution (A2) ").Compare with the concentration of the compound (XII) that detects from (XII) contrast solution (A2), the concentration of the compound (XII) that detects from (XII) metabolism solution (A2) is lower.Detect the peak from (XII) metabolism solution (A2) in addition, it does not detect from (XII) contrast solution (A2).The peak elution time of the compound of the described compound (XII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (2) (XII) metabolism solution (A1) matches.

(2) metabolism of the compound (XII) by protein of the present invention (A3)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 4 (2) in embodiment 12 (2), according to embodiment 41 (2) described methods preparations with keep reaction solns.Be similar to embodiment 41 (2), the various reaction solns after keeping with ethyl acetate extraction, and the resistates that obtains is dissolved in the 100 μ l50mM potassium phosphate buffers (pH7.0).Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 12 (2) is called " (XII) metabolism solution (A3) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 7 (2) is called " (XII) contrast solution (A3) ").Compare with the concentration of the compound (XII) that detects from (XII) contrast solution (A3), the concentration of the compound (XII) that detects from (XII) metabolism solution (A3) is lower.Detect the peak from (XII) metabolism solution (A3) in addition, it does not detect from (XII) contrast solution (A3).The peak elution time of the compound of the described compound (XII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (2) (XII) metabolism solution (A1) matches.

(3) metabolism of the compound (XII) by protein of the present invention (A10)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 4 (2) in embodiment 10 (2), according to embodiment 41 (2) described methods preparations with keep reaction solns.Be similar to embodiment 41 (2), the various reaction solns after keeping with ethyl acetate extraction, and the resistates that obtains is dissolved in the 100 μ l50mM potassium phosphate buffers (pH7.0).Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 10 (2) is called " (XII) metabolism solution (A10) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 12 (3) is called " (XII) contrast solution (A10) ").Compare with the concentration of the compound (XII) that detects from (XII) contrast solution (A10), the concentration of the compound (XII) that detects from (XII) metabolism solution (A10) is lower.Detect the peak from (XII) metabolism solution (A10) in addition, it does not detect from (XII) contrast solution (A10).The peak elution time of the compound of the described compound (XII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (2) (XII) metabolism solution (A1) matches.

(4) metabolism of the compound (XII) by protein of the present invention (A12)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 4 (2) in embodiment 34 (2), according to embodiment 41 (2) described methods preparations with keep reaction solns.Be similar to embodiment 41 (2), the various reaction solns after keeping with ethyl acetate extraction, and the resistates that obtains is dissolved in the 100 μ l 50mM potassium phosphate buffers (pH7.0).Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 34 (2) is called " (XII) metabolism solution (A12) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 34 (2) is called " (XII) contrast solution (A12) ").Compare with the concentration of the compound (XII) that detects from (XII) contrast solution (A12), the concentration of the compound (XII) that detects from (XII) metabolism solution (A12) is lower.Detect the peak from (XII) metabolism solution (A12) in addition, it does not detect from (XII) contrast solution (A12).The peak elution time of the compound of the described compound (XII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (2) (XII) metabolism solution (A1) matches.

(5) metabolism of the compound (XII) by protein of the present invention (A13)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 4 (2) in embodiment 36 (2), according to embodiment 41 (2) described methods preparations with keep reaction solns.Be similar to embodiment 41 (2), the various reaction solns after keeping with ethyl acetate extraction, and the resistates that obtains is dissolved in the 100 μ l 50mM potassium phosphate buffers (pH7.0).Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 36 (2) is called " (XII) metabolism solution (A13) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 36 (2) is called " (XII) contrast solution (A13) ").Compare with the concentration of the compound (XII) that detects from (XII) contrast solution (A13), the concentration of the compound (XII) that detects from (XII) metabolism solution (A13) is lower.Detect the peak from (XII) metabolism solution (A13) in addition, it does not detect from (XII) contrast solution (A13).The peak elution time of the compound of the described compound (XII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (2) (XII) metabolism solution (A1) matches.

(6) metabolism of the compound (XII) by protein of the present invention (A14)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 4 (2) in embodiment 38 (2), according to embodiment 41 (2) described methods preparations with keep reaction solns.Be similar to embodiment 41 (2), the various reaction solns after keeping with ethyl acetate extraction, and the resistates that obtains is dissolved in the 100 μ l 50mM potassium phosphate buffers (pH7.0).Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 38 (2) is called " (XII) metabolism solution (A14) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 38 (2) is called " (XII) contrast solution (A14) ").Compare with the concentration of the compound (XII) that detects from (XII) contrast solution (A14), the concentration of the compound (XII) that detects from (XII) metabolism solution (A14) is lower.Detect the peak from (XII) metabolism solution (A14) in addition, it does not detect from (XII) contrast solution (A14).The peak elution time of the compound of the described compound (XII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (2) (XII) metabolism solution (A1) matches.

(7) metabolism of the compound (XII) by protein of the present invention (A15)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 4 (2) in embodiment 40 (2), according to embodiment 41 (2) described methods preparations with keep reaction solns.Be similar to embodiment 41 (2), the various reaction solns after keeping with ethyl acetate extraction, and the resistates that obtains is dissolved in the 100 μ l 50mM potassium phosphate buffers (pH7.0).Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 40 (2) is called " (XII) metabolism solution (A15) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 40 (2) is called " (XII) contrast solution (A15) ").Compare with the concentration of the compound (XII) that detects from (XII) contrast solution (A15), the concentration of the compound (XII) that detects from (XII) metabolism solution (A15) is lower.Detect the peak from (XII) metabolism solution (A15) in addition, it does not detect from (XII) contrast solution (A15).The peak elution time of the compound of the described compound (XII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (2) (XII) metabolism solution (A1) matches.

(8) metabolism of the compound (XIII) by protein of the present invention (A2)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 4 (2) in embodiment 7 (2), according to embodiment 41 (3) described methods preparations with keep reaction solns.Be similar to embodiment 41 (2), the various reaction solns after keeping with ethyl acetate extraction, and the resistates that obtains is dissolved in the 100 μ l methyl-sulphoxides.Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 7 (2) is called " (XIII) metabolism solution (A2) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 7 (2) is called " (XIII) contrast solution (A2) ").Compare with the concentration of the compound (XIII) that detects from (XIII) contrast solution (A2), the concentration of the compound (XIII) that detects from (XIII) metabolism solution (A2) is lower.Detect the peak from (XIII) metabolism solution (A2) in addition, it does not detect from (XIII) contrast solution (A2).The peak elution time of the compound of the described compound (XIII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (3) (XIII) metabolism solution (A1) matches.

(9) metabolism of the compound (XIII) by protein of the present invention (A3)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 4 (2) in embodiment 12 (2), according to embodiment 41 (3) described methods preparations with keep reaction solns.Be similar to embodiment 41 (2), the various reaction solns after keeping with ethyl acetate extraction, and the resistates that obtains is dissolved in the 100 μ l methyl-sulphoxides.Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 12 (2) is called " (XIII) metabolism solution (A3) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 12 (2) is called " (XIII) contrast solution (A3) ").Compare with the concentration of the compound (XIII) that detects from (XIII) contrast solution (A3), the concentration of the compound (XIII) that detects from (XIII) metabolism solution (A3) is lower.Detect the peak from (XIII) metabolism solution (A3) in addition, it does not detect from (XIII) contrast solution (A3).The peak elution time of the compound of the described compound (XIII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (3) (XIII) metabolism solution (A1) matches.

(10) metabolism of the compound (XIII) by protein of the present invention (A10)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 4 (2) in embodiment 10 (2), according to embodiment 41 (3) described methods preparations with keep reaction solns.Be similar to embodiment 41 (2), the various reaction solns after keeping with ethyl acetate extraction, and the resistates that obtains is dissolved in the 100 μ l methyl-sulphoxides.Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 10 (2) is called " (XIII) metabolism solution (A10) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 10 (2) is called " (XIII) contrast solution (A10) ").Compare with the concentration of the compound (XIII) that detects from (XIII) contrast solution (A10), the concentration of the compound (XIII) that detects from (XIII) metabolism solution (A10) is lower.Detect the peak from (XIII) metabolism solution (A10) in addition, it does not detect from (XIII) contrast solution (A10).The peak elution time of the compound of the described compound (XIII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (3) (XIII) metabolism solution (A1) matches.

(11) metabolism of the compound (XIII) by protein of the present invention (A12)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 4 (2) in embodiment 34 (2), according to embodiment 41 (3) described methods preparations with keep reaction solns.Be similar to embodiment 41 (2), the various reaction solns after keeping with ethyl acetate extraction, and the resistates that obtains is dissolved in the 100 μ l methyl-sulphoxides.Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 34 (2) is called " (XIII) metabolism solution (A12) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 34 (2) is called " (XIII) contrast solution (A12) ").Compare with the concentration of the compound (XIII) that detects from (XIII) contrast solution (A12), the concentration of the compound (XIII) that detects from (XIII) metabolism solution (A12) is lower.Detect the peak from (XIII) metabolism solution (A12) in addition, it does not detect from (XIII) contrast solution (A12).The peak elution time of the compound of the described compound (XIII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (3) (XIII) metabolism solution (A1) matches.

(12) metabolism of the compound (XIII) by protein of the present invention (A13)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 4 (2) in embodiment 36 (2), according to embodiment 41 (3) described methods preparations with keep reaction solns.Be similar to embodiment 41 (2), the various reaction solns after keeping with ethyl acetate extraction, and the resistates that obtains is dissolved in the 100 μ l methyl-sulphoxides.Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 36 (2) is called " (XIII) metabolism solution (A13) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 36 (2) is called " (XIII) contrast solution (A13) ").Compare with the concentration of the compound (XIII) that detects from (XIII) contrast solution (A13), the concentration of the compound (XIII) that detects from (XIII) metabolism solution (A13) is lower.Detect the peak from (XIII) metabolism solution (A13) in addition, it does not detect from (XIII) contrast solution (A13).The peak elution time of the compound of the described compound (XIII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (3) (XIII) metabolism solution (A1) matches.

(13) metabolism of the compound (XIII) by protein of the present invention (A14)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 4 (2) in embodiment 38 (2), according to embodiment 41 (3) described methods preparations with keep reaction solns.Be similar to embodiment 41 (2), the various reaction solns after keeping with ethyl acetate extraction, and the resistates that obtains is dissolved in the 100 μ l methyl-sulphoxides.Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 38 (2) is called " (XIII) metabolism solution (A14) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 38 (2) is called " (XIII) contrast solution (A14) ").Compare with the concentration of the compound (XIII) that detects from (XIII) contrast solution (A14), the concentration of the compound (XIII) that detects from (XIII) metabolism solution (A14) is lower.Detect the peak from (XIII) metabolism solution (A14) in addition, it does not detect from (XIII) contrast solution (A14).The peak elution time of the compound of the described compound (XIII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (3) (XIII) metabolism solution (A1) matches.

(14) metabolism of the compound (XIII) by protein of the present invention (A15)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 4 (2) in embodiment 40 (2), according to embodiment 41 (3) described methods preparations with keep reaction solns.Be similar to embodiment 41 (2), the various reaction solns after keeping with ethyl acetate extraction, and the resistates that obtains is dissolved in the 100 μ l methyl-sulphoxides.Above-mentioned analysis condition 1 time analyzing on the HPLC solution that obtains (below, self-contained component A in the future, B component, the solution of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 40 (2) is called " (XIII) metabolism solution (A15) "; In addition, will not comprise B component from not comprising component A, the solution that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 40 (2) is called " (XIII) contrast solution (A15) ").Compare with the concentration of the compound (XIII) that detects from (XIII) contrast solution (A15), the concentration of the compound (XIII) that detects from (XIII) metabolism solution (A15) is lower.Detect the peak from (XIII) metabolism solution (A15) in addition, it does not detect from (XIII) contrast solution (A15).The peak elution time of the compound of the described compound (XIII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (3) (XIII) metabolism solution (A1) matches.

The preparation of the antibody of the present invention (A) (hereinafter referred to as " antibody of the present invention (A1) ") of embodiment 44 identification protein of the present invention (A1)

(1) preparation of the intestinal bacteria extract of expression protein of the present invention (A1)

According to embodiment 4 (2) described methods, will express the pre-overnight incubation of e. coli jm109/pKSN657F of protein of the present invention (A1), in the 1L TB substratum that comprises 50 μ g/ml penbritins, cultivate then.After recovery and ruptured cell, preparation supernatant liquor fraction (intestinal bacteria pKSN657F extract) from the cell pyrolysis liquid that obtains.

(2) purifying of protein of the present invention (A1)

Carry out Hiload HiLoad26/10Q Sepharose HP post and Bio-ScaleCeramic hydroxylapatite then successively by the supernatant liquor fraction (intestinal bacteria pKSN657F extract) that will in embodiment 44 (1), obtain, I type post CHT10-1 post is according to embodiment 2 (4) described method purifying protein of the present invention (A1).Analyzing the fraction of purifying on 10%-20%SDS-PAGE, only is the fraction of protein of the present invention (A1) to confirm that those are.

(3) preparation of antibody of the present invention (A1)

The protein of the present invention (A1) that will prepare in embodiment 44 (2) is dissolved in the 0.05M potassium phosphate buffer (pH7.0), so that concentration is 1mg/ml.With in the solution that 40 microlitres (the 40 μ l) RAS of 42 ℃ of-43 ℃ of incubations (MPL (single phosphoryl fat A)+TDM (trehalose synthesis Dicorynomycolate)+CWS (cell wall skeleton) adjuvant system (Sigma Company)) adds and thorough mixing obtains to 2ml.Respectively the mixture that obtains is administered on the New Zealand white rabbit (female, 14 ages in week, average 2.4kg) with every rabbit 1ml.Therefore, 100 μ l are subcutaneously injected into 10 positions, back.After per 3 weeks and 5 weeks, use about 1/2 of the dosage first time.At this time durations, measure antibody titer by ear vein blood sampling from rabbit.Because antibody titer increases after administration for the third time, with the immune rabbit in 2 weeks after the administration for the third time by from the neck bloodletting.The blood that obtains is added Separapit Tube (Sekisui Chemical Company), in 37 ℃ of following incubations 2 hours and centrifugal then (3000rpm, 20 minutes, room temperature).Obtain antiserum(antisera) (comprising antibody of the present invention (A1)) by reclaiming supernatant liquor.

Embodiment 45 detects protein of the present invention by antibody of the present invention (A1) and detects and express the proteinic cell of the present invention

By using the antibody of the present invention (A1) that in embodiment 44, obtains that each intestinal bacteria extract is carried out immunoblotting.The sds polyacrylamide gel electrophoresis (40mA, 1 hour) that following extract is arranged: the intestinal bacteria pKSN657F extract (comprise about 0.5pmol protein of the present invention (A1), comprise about 0.78mg protein) that in embodiment 4 (2), obtains; The intestinal bacteria pKSN2 extract (comprising about 0.78mg protein) that in embodiment 4 (2), obtains; The intestinal bacteria pKSN923F extract (comprising about 2pmol protein of the present invention (A2)) that in embodiment 7 (2), obtains; The intestinal bacteria pKSN671F extract (comprising about 2pmol protein of the present invention (A3)) that in embodiment 12 (2), obtains; The intestinal bacteria pKSN646F extract (comprising about 2pmol protein of the present invention (A4)) that in embodiment 27 (2), obtains; The intestinal bacteria pKSN11796F extract (comprising about 2pmol protein of the present invention (A10)) that in embodiment 10 (2), obtains; The intestinal bacteria pKSNSCA extract (comprising about 2pmol protein of the present invention (A9)) that in embodiment 14 (2), obtains; The intestinal bacteria pKSN849AF extract (comprising about 2pmol protein of the present invention (A11)) that in embodiment 32 (2), obtains; The intestinal bacteria pKSN1618F extract (comprising about 2pmol protein of the present invention (A12)) that in embodiment 34 (2), obtains; The intestinal bacteria pKSN474F extract (comprising about 2pmol protein of the present invention (A13)) that in embodiment 36 (2), obtains; The intestinal bacteria pKSN1491AF extract (comprising about 2pmol protein of the present invention (A14)) that in embodiment 38 (2), obtains; With the intestinal bacteria pKSN1555AF extract (comprising about 2pmol protein of the present invention (A15)) that in embodiment 40 (2), obtains.Pvdf membrane is placed on the gel.By handling 2 hours at 4 ℃ of following 30V, under the condition that is immersed in transfering buffering liquid (25mM Tris, 192mM glycine, 10% methyl alcohol), the protein transduction in the gel is moved on on the pvdf membrane simultaneously with the BioRad blotter.Using TBS+ polysorbas20 solution (50mMTris-HCl (pH 7.5), 200mM NaCl, 0.05% polysorbas20) after the washing, with the pvdf membrane that obtains incubation 30 minutes in comprising the TBS+ polysorbas20 solution of 3%BSA, be used for then in comprising the TBS+ polysorbas20 solution of 3%BSA, reacting 30 minutes with the above-mentioned antiserum(antisera) of 30,000 times of dilutions.After reaction, use TBS+ polysorbas20 solution washing pvdf membrane 2 times.Then pvdf membrane is used for reacting 30 minutes at the anti-rabbit igg goat antiserum(antisera) with alkaline phosphatase (Santa Cruz Biotechnology Company) mark of the TBS+ polysorbas20 solution that comprises 3%BSA with 3000 times of dilutions.After reaction, with TBS+ polysorbas20 solution washing pvdf membrane 2 times be immersed in the NBT-BCIP solution (Sigma Company).Detect corresponding to protein of the present invention (A1), (A2), (A3), (A4), (A11), (A12), (A13), (A14) and (A15) and protein of the present invention (A9) and (A10) in each the dyeing of band.The reagent that is used in the intestinal bacteria pKSN2 extract (comprising about 0.78mg protein) that obtains among the embodiment 4 (2) does not detect the dyeing band.

Embodiment 46 wherein codon uses at preparation and the expression of expressing the DNA of the present invention (A1) (hereinafter referred to as " DNA of the present invention (A1) S ") that adjusts in soybean

(1) preparation of DNA of the present invention (A1) S

Have the primer and primer of nucleotide sequence shown in the SEQ ID NO:192 by use, carry out PCR with Pyrobest archaeal dna polymerase (Takara Shuzo Company) according to incidental handbook with nucleotide sequence shown in the SEQ IDNO:213.With the aliquot of the PCR product that obtains as using primer similarly and having the template of the PCR that the primer of nucleotide sequence shown in the SEQ IDNO:212 carries out with nucleotide sequence shown in the SEQ ID NO:191.In addition, with the aliquot of PCR product as using primer similarly and having the template of the PCR that the primer of nucleotide sequence shown in the SEQ ID NO:211 carries out with nucleotide sequence shown in the SEQ ID NO:190.The reaction soln that obtains is called reaction soln 1.

According to incidental handbook, have the primer and primer of nucleotide sequence shown in the SEQ ID NO:195 with nucleotide sequence shown in the SEQ ID NO:210 by use, (Takara Shuzo Company) carries out PCR with the Pyrobest archaeal dna polymerase.With the aliquot of the PCR product that obtains as using primer similarly and having the template of the PCR that the primer of nucleotide sequence shown in the SEQ IDNO:209 carries out with nucleotide sequence shown in the SEQ ID NO:194.In addition, with the aliquot of PCR product as using primer similarly and having the template of the PCR that the primer of nucleotide sequence shown in the SEQ ID NO:208 carries out with nucleotide sequence shown in the SEQ ID NO:193.The reaction soln that obtains is called reaction soln 2.

According to incidental handbook, have the primer and primer of nucleotide sequence shown in the SEQ ID NO:198 with nucleotide sequence shown in the SEQ ID NO:207 by use, (Takara Shuzo Company) carries out PCR with the Pyrobest archaeal dna polymerase.With the aliquot of the PCR product that obtains as using primer similarly and having the template of the PCR that the primer of nucleotide sequence shown in the SEQ IDNO:206 carries out with nucleotide sequence shown in the SEQ ID NO:197.In addition, with the aliquot of PCR product as using primer similarly and having the template of the PCR that the primer of nucleotide sequence shown in the SEQ ID NO:205 carries out with nucleotide sequence shown in the SEQ ID NO:196.The reaction soln that obtains is called reaction soln 3.

According to incidental handbook, have the primer and primer of nucleotide sequence shown in the SEQ ID NO:201 with nucleotide sequence shown in the SEQ ID NO:204 by use, (Takara Shuzo Company) carries out PCR with the Pyrobest archaeal dna polymerase.With the aliquot of the PCR product that obtains as using primer similarly and having the template of the PCR that the primer of nucleotide sequence shown in the SEQ IDNO:203 carries out with nucleotide sequence shown in the SEQ ID NO:200.In addition, with the aliquot of PCR product as using primer similarly and having the template of the PCR that the primer of nucleotide sequence shown in the SEQ ID NO:202 carries out with nucleotide sequence shown in the SEQ ID NO:199.The reaction soln that obtains is called reaction soln 4.

The reaction soln 1 to 4 that so obtains is mixed.According to incidental handbook, by the aliquot of its mixture is had the primer and the primer with nucleotide sequence shown in the SEQ ID NO:202 of nucleotide sequence shown in the SEQ ID NO:190 as template and use, (Takara Shuzo Company) carries out PCR with the Pyrobest archaeal dna polymerase.Confirm the nucleotide sequence of the DNA of amplification.Acquisition has wherein, and nucleotide sequence 5 '-cat-3 ' is connected to 5 ' terminal upstream of nucleotide sequence shown in the SEQ ID NO:214 and the DNA that nucleotide sequence 5 '-aagctt-3 ' is connected to the sequence in 3 ' terminal downstream of nucleotide sequence shown in the SEQ ID NO:214.

The codon that shows the DNA of the present invention (A1) with nucleotide sequence shown in the SEQ ID NO:6 in table 22 and table 23 uses (GC content 70.58%).The codon that shows soybean in table 24 and table 25 uses (GC content 46.12%, the codon of being published by Kazusa DNA research institute uses database (http://www.kazusa.or.jp/codon)).The codon that shows the DNA of the present invention (A1) with nucleotide sequence shown in the SEQ ID NO:214 in table 26 and table 27 uses (51.59% GC content).

Table 22

Codon	％	Codon	％
Codon	％	Codon	％	TTT	0.00	TCT	0.00
TTC	3.18	TCC	1.71	TTT	0.00	TCT	0.00
TTC	3.18	TCC	1.71	TTA	0.00	TCA	0.00
TTG	1.22	TCG	2.20	TTA	0.00	TCA	0.00
TTG	1.22	TCG	2.20	CTT	0.00	CCT	0.00
CTC	3.67	CCC	4.16	CTT	0.00	CCT	0.00
CTC	3.67	CCC	4.16	CTA	0.00	CCA	0.00
CTG	7.09	CCG	2.69	CTA	0.00	CCA	0.00
CTG	7.09	CCG	2.69	ATT	0.24	ACT	0.24
ATC	4.16	ACC	2.69	ATT	0.24	ACT	0.24
ATC	4.16	ACC	2.69	ATA	0.00	ACA	0.24
ATG	2.69	ACG	1.96	ATA	0.00	ACA	0.24
ATG	2.69	ACG	1.96	GTT	0.24	GCT	0.00
GTC	3.67	GCC	7.58	GTT	0.24	GCT	0.00
GTC	3.67	GCC	7.58	GTA	0.00	GCA	0.49
GTG	3.18	GCG	3.42	GTA	0.00	GCA	0.49

Table 23

Codon	％	Codon	％
Codon	％	Codon	％	TAT	0.00	TGT	0.24
TAC	1.47	TGC	0.98	TAT	0.00	TGT	0.24
TAC	1.47	TGC	0.98	TAA	0.00	TGA	0.00
TAG	0.24	TGG	0.98	TAA	0.00	TGA	0.00
TAG	0.24	TGG	0.98	CAT	0.24	CGT	1.22
CAC	2.20	CGC	4.40	CAT	0.24	CGT	1.22
CAC	2.20	CGC	4.40	CAA	0.24	CGA	0.24
CAG	2.93	CGG	4.16	CAA	0.24	CGA	0.24

AAT	0.00	AGT	0.00
AAT	0.00	AGT	0.00	AAC	1.22	AGC	0.49
AAA	0.24	AGA	0.00	AAC	1.22	AGC	0.49
AAA	0.24	AGA	0.00	AAG	0.98	AGG	0.00
GAT	0.98	GGT	0.98	AAG	0.98	AGG	0.00
GAT	0.98	GGT	0.98	GAC	7.82	GGC	3.42
GAA	0.73	GGA	0.24	GAC	7.82	GGC	3.42
GAA	0.73	GGA	0.24	GAG	5.38	GGG	1.22

Table 24

Codon	％	Codon	％
Codon	％	Codon	％	TTT	2.03	TCT	1.71
TTC	2.09	TCC	1.21	TTT	2.03	TCT	1.71
TTC	2.09	TCC	1.21	TTA	0.82	TCA	1.45
TTG	2.21	TCG	0.44	TTA	0.82	TCA	1.45
TTG	2.21	TCG	0.44	CTT	2.36	CCT	2.00
CTC	1.66	CCC	1.01	CTT	2.36	CCT	2.00
CTC	1.66	CCC	1.01	CTA	0.82	CCA	2.05
CTG	1.22	CCG	0.40	CTA	0.82	CCA	2.05
CTG	1.22	CCG	0.40	ATT	2.61	ACT	1.78
ATC	1.64	ACC	1.49	ATT	2.61	ACT	1.78
ATC	1.64	ACC	1.49	ATA	1.27	ACA	1.51
ATG	2.27	ACG	0.41	ATA	1.27	ACA	1.51
ATG	2.27	ACG	0.41	GTT	2.67	GCT	2.81
GTC	1.24	GCC	1.69	GTT	2.67	GCT	2.81
GTC	1.24	GCC	1.69	GTA	0.73	GCA	2.27
GTG	2.20	GCG	0.59	GTA	0.73	GCA	2.27

Table 25

Codon	％	Codon	％
Codon	％	Codon	％	TAT	1.61	TGT	0.72
TAC	1.53	TGC	0.75	TAT	1.61	TGT	0.72
TAC	1.53	TGC	0.75	TAA	0.11	TGA	0.09
TAG	0.06	TGG	1.21	TAA	0.11	TGA	0.09
TAG	0.06	TGG	1.21	CAT	1.33	CGT	0.72
CAC	1.09	CGC	0.63	CAT	1.33	CGT	0.72
CAC	1.09	CGC	0.63	CAA	2.04	CGA	0.38
CAG	1.71	CGG	0.27	CAA	2.04	CGA	0.38
CAG	1.71	CGG	0.27	AAT	2.10	AGT	1.21
AAC	2.27	AGC	1.08	AAT	2.10	AGT	1.21
AAC	2.27	AGC	1.08	AAA	2.63	AGA	1.42
AAG	3.83	AGG	1.35	AAA	2.63	AGA	1.42
AAG	3.83	AGG	1.35	GAT	3.29	GGT	2.17
GAC	2.06	GGC	1.38	GAT	3.29	GGT	2.17
GAC	2.06	GGC	1.38	GAA	3.35	GGA	2.23
GAG	3.46	GGG	1.29	GAA	3.35	GGA	2.23

Table 26

Codon	％	Codon	％
Codon	％	Codon	％	TTT	1.71	TCT	0.98
TTC	1.47	TCC	0.73	TTT	1.71	TCT	0.98
TTC	1.47	TCC	0.73	TTA	0.98	TCA	0.98
TTG	2.93	TCG	0.24	TTA	0.98	TCA	0.98
TTG	2.93	TCG	0.24	CTT	3.18	CCT	2.44
CTC	2.20	CCC	1.22	CTT	3.18	CCT	2.44
CTC	2.20	CCC	1.22	CTA	0.98	CCA	2.69
CTG	1.71	CCG	0.49	CTA	0.98	CCA	2.69

ATT	2.20	ACT	1.71
ATT	2.20	ACT	1.71	ATC	1.22	ACC	1.47
ATA	0.98	ACA	1.47	ATC	1.22	ACC	1.47
ATA	0.98	ACA	1.47	ATG	2.69	ACG	0.49
GTT	2.93	GCT	4.16	ATG	2.69	ACG	0.49
GTT	2.93	GCT	4.16	GTC	1.22	GCC	2.69
GTA	0.73	GCA	3.67	GTC	1.22	GCC	2.69
GTA	0.73	GCA	3.67	GTG	2.20	GCG	0.98

Table 27

Codon	％	Codon	％
Codon	％	Codon	％	TAT	0.73	TGT	0.73
TAC	0.73	TGC	0.49	TAT	0.73	TGT	0.73
TAC	0.73	TGC	0.49	TAA	0.00	TGA	0.00
TAG	0.24	TGG	0.98	TAA	0.00	TGA	0.00
TAG	0.24	TGG	0.98	CAT	1.47	CGT	1.47
CAC	0.98	CGC	1.47	CAT	1.47	CGT	1.47
CAC	0.98	CGC	1.47	CAA	1.71	CGA	0.73
CAG	1.47	CGG	0.49	CAA	1.71	CGA	0.73
CAG	1.47	CGG	0.49	AAT	0.73	AGT	0.73
AAC	0.49	AGC	0.73	AAT	0.73	AGT	0.73
AAC	0.49	AGC	0.73	AAA	0.49	AGA	2.93
AAG	0.73	AGG	2.93	AAA	0.49	AGA	2.93
AAG	0.73	AGG	2.93	GAT	5.38	GGT	1.71
GAC	3.42	GGC	1.22	GAT	5.38	GGT	1.71
GAC	3.42	GGC	1.22	GAA	2.69	GGA	1.96
GAG	3.42	GGG	0.98	GAA	2.69	GGA	1.96

(2) has the production of the transformed into escherichia coli of protein of the present invention (A1) S

The DNA with nucleotide sequence shown in the SEQ IDNO:214 of acquisition in embodiment 46 (1) with restriction enzyme NdeI and HindIII digestion.To connect NdeI site and the plasmid between the HindIII site (hereinafter referred to as " pKSN657soy ") that inserts pKSN2 with the DNA that obtains wherein to have nucleotide sequence shown in the SEQ ID NO:214 with the DNA of the acquisition of NdeI and HindIII digestion and plasmid pKSN2.Described plasmid is imported e. coli jm109.The intestinal bacteria transformant that obtains is called JM109/pKSN657soy.

(3) expression and the described recovery of protein of protein of the present invention (A1) in intestinal bacteria

Be similar to embodiment 4 (2), cultivate in e. coli jm109/pKSN657soy that in embodiment 46 (2), obtains and the e. coli jm109/pKSN657 that in embodiment 4 (1), obtains each.Reclaim cell.The preparation cell pyrolysis liquid.Under embodiment 4 (2) described methods, preparation supernatant liquor fraction from cell pyrolysis liquid (below, will be called " intestinal bacteria pKSN657soy extract " from the supernatant liquor fraction that e. coli jm109/pKSN657soy obtains and will be called " e. coli jm109/pKSN657 extract " from the supernatant liquor fraction that e. coli jm109/pKSN657 obtains).The amount of the P450 of each the proteinic amount that comprises in intestinal bacteria pKSN657soy extract can be comparable to and be higher than the amount of the P450 of each the proteinic amount that comprises in intestinal bacteria pKSN657 extract.

Embodiment 47 imports plant with DNA of the present invention (A1) S

(1) is used for the directly structure-part 1 of the chloroplast expression plasmid that comprises DNA of the present invention (A1) S of importing

The plasmid that structure comprises chimeric DNA is as importing the plasmid of plant with particle bombardment with DNA of the present invention (A1) S, and DNA of the present invention (A1) S is right after after the nucleotide sequence of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and does not have a variation of codon frame in the described chimeric DNA.

At first, the DNA that comprises nucleotide sequence shown in the SEQ ID NO:214 by pcr amplification.By the pKSN657soy that will in embodiment 46 (2), obtain as template with by the oligonucleotide that will form by nucleotide sequence shown in the SEQ IDNO:394 be used as primer by the oligonucleotide that nucleotide sequence shown in the SEQ ID NO:395 is formed and carry out PCR.PCR uses KOD-plus (ToyoboCompany).According to the following PCR that carries out: under 94 ℃, kept 2 minutes; 30 circulations, circulation comprise keep 94 ℃ 30 seconds, then 50 ℃ 30 seconds, then 68 ℃ 60 seconds; Remain at last 68 ℃ 30 seconds.By carrying out program MagExtractor-PCR﹠amp according to subsidiary handbook; Gel-Clean up (Toyobo Company) reclaims the DNA and the purifying of amplification.After DNA, reclaim the DNA that comprises nucleotide sequence shown in the SEQID NO:214 with restriction enzyme EcoT22I and SacI digestion purifying.After the plasmid pUCrSt657 that in embodiment 16 (2), obtains with restriction enzyme EcoT22I and SacI digestion, the DNA that separates about 2.9kbp, it has the nucleotide sequence that derives from pUC19 and the sequence of soybean (cv.Jack) the RuBPC small subunit chloroplast transit peptides of encoding.The DNA that obtains is connected the pUCrSt657soy (Figure 48) that comprises chimeric DNA with acquisition with the above-mentioned DNA that comprises nucleotide sequence shown in the SEQ ID NO:214, DNA wherein of the present invention (A1) S is right after after the nucleotide sequence of soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides of encoding and does not have a variation of codon frame.

Digest the plasmid pUCrSt657soy of acquisition to separate the DNA that comprises nucleotide sequence shown in the SEQ ID NO:214 with restriction enzyme BamHI and SacI.Described DNA is inserted between the BglII restriction enzyme sites of the plasmid pNdG6-Δ T that obtain among the embodiment 16 (2) and the SacI restriction enzyme sites to obtain plasmid pSUM-NdG6-rSt-657soy (Figure 49), wherein the CR16G6 promotor has been connected to the downstream of chimeric DNA, and DNA of the present invention (A1) S is right after after the nucleotide sequence of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and does not have a variation of codon frame in the described chimeric DNA.

Next, plasmid is imported bacillus coli DH 5 alpha competent cell (Takara ShuzoCompany) and selection amicillin resistance cell.In addition, by using BigDye to stop the nucleotide sequence that cycle sequencing easy reaction test kit 3.0 editions (PE Applied Biosystems Company) and dna sequencing instrument 3100 (PE Applied Biosystems Company) mensuration are included in the plasmid in the selected amicillin resistance bacterial strain.As a result, confirm that plasmid pSUM-NdG6-rSt-657soy has nucleotide sequence shown in the SEQ ID NO:214.

(2) be used for directly structure-partly (2) of the chloroplast expression plasmid with DNA of the present invention (A1) S of importing

Structure imports DNA of the present invention (A1) S with particle bombardment the plasmid of plant.This plasmid comprises chimeric DNA, and DNA wherein of the present invention (A1) S is right after after 12 amino acid whose nucleotide sequences of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and the mature protein thereafter of encoding and does not have a variation of codon frame.At first, the DNA that comprises nucleotide sequence shown in the SEQ ID NO:214 by pcr amplification.By the pKSN657soy that will in embodiment 46 (2), obtain as template with by the oligonucleotide that will form by nucleotide sequence shown in the SEQ ID NO:395 be used as primer by the oligonucleotide that nucleotide sequence shown in the SEQ ID NO:396 is formed and carry out PCR.PCR uses KOD-plus (Toyobo Company).According to the following PCR that carries out: under 94 ℃, kept 2 minutes; 25 circulations, circulation comprise keep 94 ℃ 30 seconds, then 46 ℃ 30 seconds, then 68 ℃ 60 seconds; Remain at last 68 ℃ 3 minutes.By carrying out program MagExtractor-PCR﹠amp according to subsidiary handbook; Gel-Clean up (Toyobo Company) reclaims the DNA and the purifying of amplification.After DNA, reclaim the DNA that comprises nucleotide sequence shown in the SEQ ID NO:214 with restriction enzyme SacI digestion purifying.

Plasmid pKFrSt12-657 with restriction enzyme BspHI digestion acquisition in embodiment 16 (3).Then by use TaKaRa BKL test kit (TaKaRa ShuzoCompany) according to subsidiary handbook with the flat endization of DNA with 5 ' terminal dephosphorylation.Next, after with restriction enzyme SacI dna digestion, separate the DNA that derives from plasmid pKFrSt12.With described DNA be connected with the DNA that comprises nucleotide sequence shown in the SEQ ID NO:214 with SacI digestion, so that obtain to comprise the plasmid pKFrSt12-657soy (Figure 50) of chimeric DNA, DNA of the present invention (A1) S is right after after 12 amino acid whose nucleotide sequences of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and the mature protein thereafter of encoding and does not have a variation of codon frame in described chimeric DNA.

Digest the plasmid pKFrSt12-657soy of acquisition to separate the DNA that comprises nucleotide sequence shown in the SEQ ID NO:214 with restriction enzyme BamHI and SacI.Described DNA is inserted between the BglII restriction enzyme sites of plasmid pNdG6-Δ T and the SacI restriction enzyme sites to obtain plasmid pSUM-NdG6-rSt12-657soy (Figure 51), wherein the CR16G6 promotor has been connected to the downstream of chimeric DNA, and DNA described in the described chimeric DNA is right after after the nucleotide sequence of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and does not have a variation of codon frame.

Next, plasmid is imported bacillus coli DH 5 alpha competent cell (Takara ShuzoCompany) and selection amicillin resistance cell.In addition, by using BigDye to stop the nucleotide sequence that cycle sequencing easy reaction test kit 3.0 editions (PE Applied Biosystems Company) and dna sequencing instrument 3100 (PE Applied Biosystems Company) mensuration are included in the plasmid in the amicillin resistance bacterial strain.As a result, confirm that plasmid pSUM-NdG6-rSt12-657soy has nucleotide sequence shown in the SEQ ID NO:214.

(3) DNA of the present invention (A1) S is imported soybean

Except using B5 medium (O.L.Gamborg etc., Exp.Cell Res. (1986) 50p151) VITAMIN source substitutes beyond the VITAMIN source of MS substratum, prepares the globular embryo of soybean (Cultivar: Fayette and Jack) according to embodiment 17 (1) described methods.

Be transplanted to the globular embryo that obtains in the fresh body somatic embryo growth medium and cultivated 2-3 days.According to embodiment 17 (2) described methods, plasmid pSUM-NdG6-rSt-657soy that will make up in embodiment 47 (1) or the plasmid pSUM-NdG6-rSt12-657soy that makes up in embodiment 47 (2) import described globular embryo.

(4) with Totomycin selective body somatic embryo

Except the VITAMIN source with B5 medium substitutes the VITAMIN source of MS substratum, carry out being chosen in the globular embryo after gene imports that obtains among the embodiment 47 (3) with Totomycin according to embodiment 17 (3) described methods.Yet, after transplanting for the second time, will add 0.2 (w/v) % take off the substratum of acetyl gellan gum or do not add take off the acetyl gellan gum liquid nutrient medium as somatic embryo selection substratum.In the situation of liquid medium within, cultivation has the rotation that per minute 90 eases up.

(5) with compound (II) selective body somatic embryo

Except the VITAMIN source with B5 medium substitutes the VITAMIN source of MS substratum, be chosen in the later globular embryo of quiding gene that obtains among the embodiment 47 (3) according to embodiment 17 (4) described method compounds (II).

(6), comply with and cultivate from the plant regeneration of somatic embryo

According to embodiment 17 (5) described methods, carry out the regeneration of plant from the globular embryo of embodiment 47 (4) or 47 (5), selecting.Yet, the agar concentration of growing in the substratum is adjusted to 0.8 (w/v) % or 1.0 (w/v) %.In addition, substitute the VITAMIN source of the MS substratum of germination substratum with the VITAMIN source of B5 medium.

The plant of correspondingly using embodiment 17 (6) described methods will have the leaf of root and growth complies with and cultivates and gathers in the crops.

(7) resistance of herbicidal compound (II) is assessed

Be evaluated at the resistance level of the aftergrowth of acquisition among the embodiment 47 (6) according to embodiment 17 (4) described methods to compound (II).

(8) be used for the structure of the chloroplast expression plasmid that Agrobacterium imports with DNA of the present invention (A1) S

Structure imports DNA of the present invention (A1) S with agrobacterium co-cultivation the plasmid of plant.Obtaining chimeric DNA, DNA wherein of the present invention (A1) S is right after after the nucleotide sequence of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and does not have a variation of codon frame with restriction enzyme NotI digested plasmid pSUM-NdG6-rSt-657soy.Described DNA is inserted among the embodiment 18 in the NotI restriction site of the above-mentioned binary plasmid carrier pBI121S that obtains to obtain plasmid pBI-NdG6-rSt-657soy (Figure 52).In addition, separating chimeric DNA, DNA wherein of the present invention (A1) S is right after after 12 amino acid whose nucleotide sequences of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and the mature protein thereafter of encoding and does not have a variation of codon frame with restriction enzyme NotI digested plasmid pSUM-NdG6-rSt12-657soy.This DNA is inserted in the NotI restriction site of above-mentioned binary plasmid carrier pBI121S to obtain plasmid pBI-NdG6-rSt12-657soy (Figure 53).

(9) DNA of the present invention (A1) S is imported tobacco

Plasmid pBI-NdG6-rSt-657soy and pBI-NdG6-rSt12-657soy that use obtains in embodiment 47 (8) import tobacco with agrobacterium co-cultivation with DNA of the present invention (A1) S.

At first, according to embodiment 19 described methods, respectively plasmid pBI-NdG6-rSt-657soy and pBI-NdG6-rSt12-657soy are imported agrobacterium tumefaciens lba4404 (Clontech Company).Separate transgenosis Agrobacterium with pBI-NdG6-rSt-657soy or pBI-NdG6-rSt12-657soy.

Then, except in comprising the LB liquid nutrient medium of 25mg/L kantlex 30 ℃ of following incubated overnight have the transgenosis Agrobacterium of above-mentioned plasmid, according to embodiment 19 described methods described Agrobacterium is used for gene is imported tobacco.The transgene tobacco in the T-DNA zone of pBI-NdG6-rSt-657soy or pBI-NdG6-rSt12-657soy has been integrated in acquisition respectively.

(10) use the resistance of the blade of DNA of the present invention (A1) S transgene tobacco to assess

Gather leaf from the transgene tobacco that 35 strains obtain among embodiment 47 (9).Every leaf is divided into several, and wherein every 5-7mm is wide.Blade planted contains 0,0.05,0.1 or the MS nutrient agar of 0.2mg/L compound (II) on and in illumination, at room temperature cultivate.At the 11st day that cultivates, observe the weeding damage of each blade.In addition, blade planted contains 0,0.01,0.02,0.05 or the MS nutrient agar of 0.1mg/L compound (XII) on and in illumination, at room temperature cultivate.At the 7th day that cultivates, observe the weeding damage of each blade.In contrast, do not carry out the tobacco leaf that gene imports (below, be called " wild-type tobacco ") with 20 and be used for each concentration.By to the blade of continuous growth note 1 minute,, measure average score to every group to the half withered blade note 0.5 minute of wherein observing chemical damage with to bleaching and withered blade note 0 minute.For in compound (II) and the compound (XII) each, the tobacco leaf that DNA of the present invention (A1) S (the T-DNA zone of plasmid pBI-NdG6-rSt-657soy or pBI-NdG6-rSt12-657soy) has imported provides the mark higher than wild-type tobacco.

Embodiment 48 obtains DNA of the present invention (A16)

(1) preparation of the chromosomal DNA of decorative chains mould IFO 13069t

According to embodiment 31 (1) described methods, the chromosomal DNA of preparation decorative chains mould IFO 13069t.

According to embodiment 29 described methods, by will in embodiment 48 (1), carrying out PCR as template with by the use primer to 14 by the chromosomal DNA from decorative chains mould IFO13069t preparation.Be similar to embodiment 31 (2), with the amplification dna clone to cloning vector pCRII-TOPO (InvitrogenCompany).Analyze its sequence.As a result, be provided at represented nucleotide sequence in the Nucleotide 343 to 1069 of nucleotide sequence shown in the SEQ ID NO:225.

In addition, the chromosomal DNA that in embodiment 48 (1), prepares with restriction enzyme PvuII digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:265 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:266 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1 to 501 of nucleotide sequence shown in the SEQ ID NO:235.

In addition, the chromosomal DNA that in embodiment 48 (1), prepares with restriction enzyme PvuII digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:267 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:268 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1044 to 1454 of nucleotide sequence shown in the SEQ ID NO:235.

(3) sequential analysis of DNA of the present invention (A16)

By the nucleotide sequence that the nucleotide sequence acquisition that provided by embodiment 48 (2) DNA that obtain is represented in SEQ IDNO:235 is provided.In described nucleotide sequence, there are two open reading-frame (ORF)s (ORF).Therefore, comprise by 1251 Nucleotide (comprising terminator codon) and form and the nucleotide sequence (SEQ ID NO:225) of 416 amino-acid residues of encoding (SEQ ID NO:215) and form and the nucleotide sequence (SEQ ID NO:255) of 65 amino-acid residues (the SEQ IDNO:245) that encode by 198 Nucleotide (comprising terminator codon).The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:225 (SEQ ID NO:215) it is calculated that and is 46013Da.In addition, the proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:255 (SEQ ID NO:245) it is calculated that and is 6768Da.

Embodiment 49 expression of DNA of the present invention (A16) in intestinal bacteria

(1) has the generation of the transformed into escherichia coli of DNA of the present invention (A16)

By using GeneAmp High Fidelity PCR System (Applied BiosystemsJapan Company) and in embodiment 48 (1), carrying out PCR as template from the chromosomal DNA of decorative chains mould IFO 13069t preparation by use.As primer, use oligonucleotide with nucleotide sequence shown in the SEQ ID NO:269 and pairing with oligonucleotide of nucleotide sequence shown in the SEQ ID NO:286.By adding 2 kinds every kind primer that amounts to 200nM, the above-mentioned chromosomal DNA of 50ng, the 5.0 μ l dNTP mixture (mixtures of every kind of 2.0mM of 4 kinds of dNTP; Clontech company), 5 μ l 10x damping fluids (comprise MgCl ₂) and 0.5 μ l GeneAmpHF enzyme mixture and by adding distilled water, the PCR reaction soln amounts to 50 μ l.The reaction conditions of PCR is to remain on 97 ℃ after 1 minute, repeating 10 circulations, circulation comprise remain on 97 ℃ 15 seconds, then 60 ℃ 30 seconds, then 72 ℃ 90 seconds; Carry out 15 circulations then, circulation comprise remain on 97 ℃ 15 seconds, then 60 ℃ 30 seconds, then 72 ℃ 90 seconds (wherein each circulation remains on 72 ℃ increases by 20 seconds); Keep then 72 ℃ 7 minutes.Be similar to embodiment 32 (1), purify DNA and being cloned among the cloning vector pCRII-TOPO (InvitrogenCompany) from the PCR reaction soln.By having SEQ ID NO:57 respectively, the oligonucleotide of nucleotide sequence shown in 59,267,286 and 288 is as the nucleotide sequence of the plasmid DNA of primer analysis acquisition.Based on the result who obtains, the plasmid that will have nucleotide sequence shown in the SEQ ID NO:235 is called pCR452F.Be similar to embodiment 32 (1), with restriction enzyme NdeI and HindIII digestion pCR452F.The DNA of the about 1.5kbp of purifying from digestion product.Connect with the DNA of the acquisition of NdeI and HindIII digestion and plasmid pKSN2 to obtain to comprise the plasmid of nucleotide sequence shown in the SEQ ID NO:235, wherein with between the NdeI site and HindIII site of the DNA insertion pKSN2 of code book invention protein (A16) (hereinafter referred to as " pKSN452F ").Described plasmid is imported e. coli jm109.The intestinal bacteria transformant that obtains is called JM109/pKSN452F.

(2) expression and the described recovery of protein of protein of the present invention (A16) in intestinal bacteria

Be similar to embodiment 4 (2), cultivate among e. coli jm109/pKSN452F and the JM109/pKSN2 each.Reclaim cell.The preparation cell pyrolysis liquid.Under embodiment 4 (2) described methods, preparation supernatant liquor fraction from cell pyrolysis liquid (below, to be called " intestinal bacteria pKSN452F extract " available from the supernatant liquor fraction of e. coli jm109/pKSN452F, will be called " intestinal bacteria pKSN2 extract ") available from the supernatant liquor fraction of e. coli jm109/pKSN2.

Similar embodiment 32 (3), prepare the reaction soln of 30 μ l and remain on 30 ℃ following 10 minutes.Yet,, use the supernatant liquor fraction (intestinal bacteria pKSN452F extract or intestinal bacteria pKSN2 extract) of preparation in embodiment 49 (2) as the supernatant liquor fraction.Analyze with the reaction soln after the ethyl acetate extraction maintenance and with extract layer TLC.After launching the TLC plate, check on it corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).From the reaction soln that comprises intestinal bacteria pKSN452F extract, detect spot corresponding to compound (III).Contrast does not detect this spot in the reaction soln that comprises intestinal bacteria pKSN2 extract therewith.

Embodiment 50 obtains DNA of the present invention (A17)

(1) preparation of the chromosomal DNA of streptomyces griseus ATCC 10137

According to embodiment 31 (1) described methods, the chromosomal DNA of preparation streptomyces griseus ATCC 10137.

(2) has the separation of the DNA of DNA of the present invention (A17) partial nucleotide sequence

According to embodiment 29 described methods, by will in embodiment 50 (1), carrying out PCR as template with by the use primer to 14 by the chromosomal DNA from streptomyces griseus ATCC 10137 preparations.Be similar to embodiment 31 (2), with the amplification dna clone to cloning vector pCRII-TOPO (Invitrogen Company).Analyze its nucleotide sequence.As a result, be provided at represented nucleotide sequence in the Nucleotide 343 to 1069 of nucleotide sequence shown in the SEQ ID NO:226.

In addition, the chromosomal DNA that in embodiment 50 (1), prepares with restriction enzyme SmaI digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:270 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:271 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1 to 361 of nucleotide sequence shown in the SEQ ID NO:236.

In addition, the chromosomal DNA that in embodiment 50 (1), prepares with restriction enzyme PvuII digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:272 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:273 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1035 to 1454 of nucleotide sequence shown in the SEQ ID NO:236.

(3) sequential analysis of DNA of the present invention (A17)

By the nucleotide sequence that the nucleotide sequence acquisition that provided by embodiment 50 (2) DNA that obtain is represented in SEQ IDNO:236 is provided.In described nucleotide sequence, there are two open reading-frame (ORF)s (ORF).Therefore, comprise by 1251 Nucleotide (comprising terminator codon) and form and the nucleotide sequence (SEQ ID NO:226) of 416 amino-acid residues of encoding (SEQ ID NO:216) and form and the nucleotide sequence (SEQ ID NO:256) of 65 amino-acid residues (the SEQ IDNO:246) that encode by 198 Nucleotide (comprising terminator codon).The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:226 (SEQ ID NO:216) it is calculated that and is 46082Da.The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:256 (SEQ ID NO:246) it is calculated that and is 6768Da.At the nucleotide sequence shown in the SEQ IDNO:256 with identical at nucleotide sequence 100% shown in the SEQ ID NO:255.The aminoacid sequence of representing in SEQ ID NO:246 is identical with the aminoacid sequence of representing in SEQ ID NO:245 100%.

Embodiment 51 expression of DNA of the present invention (A17) in intestinal bacteria

(1) has the generation of the transformed into escherichia coli of DNA of the present invention (A17)

Except the chromosomal DNA that uses preparation from the streptomyces griseus ATCC 10137 of embodiment 50 (1) as template and use oligonucleotide with nucleotide sequence shown in the SEQ ID NO:274 and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:275 as the primer, be similar to embodiment 32 (1) and carry out PCR.Be similar to embodiment 32 (1), purify DNA and being cloned among the cloning vector pCRII-TOPO (Invitrogen Company) from the PCR reaction soln.By having SEQ ID NO:57 respectively, the oligonucleotide of nucleotide sequence shown in 59,274,276 and 277 is as the nucleotide sequence of the plasmid DNA of primer order-checking acquisition.Based on the result who obtains, the plasmid that will have nucleotide sequence shown in the SEQ ID NO:236 is called pCR608F.Be similar to embodiment 32 (1), with restriction enzyme NdeI and HindIII digestion pCR608F.The DNA of the about 1.5kbp of purifying from digestion product.Connect with the DNA of the acquisition of NdeI and HindIII digestion and plasmid pKSN2 to obtain to comprise the plasmid of nucleotide sequence shown in the SEQ ID NO:236, wherein with between the NdeI site and HindIII site of the DNA insertion pKSN2 of code book invention protein (A17) (hereinafter referred to as " pKSN608F ").Described plasmid is imported e. coli jm109.The intestinal bacteria transformant that obtains is called JM109/pKSN608F.

(2) expression and the described recovery of protein of protein of the present invention (A17) in intestinal bacteria

Be similar to embodiment 4 (2), cultivate among e. coli jm109/pKSN608F and the JM109/pKSN2 each.Reclaim cell.The preparation cell pyrolysis liquid.Under embodiment 4 (2) described methods, preparation supernatant liquor fraction from cell pyrolysis liquid (below, to be called " intestinal bacteria pKSN608F extract " available from the supernatant liquor fraction of e. coli jm109/pKSN608F, will be called " intestinal bacteria pKSN2 extract ") available from the supernatant liquor fraction of e. coli jm109/pKSN2.

Be similar to embodiment 32 (3), prepare the reaction soln of 30 μ l and remain on 30 ℃ following 10 minutes.Yet,, use the supernatant liquor fraction (intestinal bacteria pKSN608F extract or intestinal bacteria pKSN2 extract) of preparation in embodiment 51 (2) as the supernatant liquor fraction.Analyze with the reaction soln after the ethyl acetate extraction maintenance and with extract layer TLC.After launching the TLC plate, check on it corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).From the reaction soln that comprises intestinal bacteria pKSN608F extract, detect spot corresponding to compound (III).Contrast does not detect this spot in the reaction soln that comprises intestinal bacteria pKSN2 extract therewith.

Embodiment 52 obtains DNA of the present invention (A18)

According to embodiment 31 (1) described methods, the chromosomal DNA of look streptomycete IFO 12735 is not produced in preparation.

(2) has the separation of the DNA of DNA of the present invention (A18) partial nucleotide sequence

According to embodiment 29 described methods, carry out PCR as template with by the use primer to 17 by the chromosomal DNA that does not produce look streptomycete IFO 12735 that will in embodiment 52 (1), prepare.Be similar to embodiment 31 (2), with the amplification dna clone to cloning vector pCRII-TOPO (Invitrogen Company).Analyze its nucleotide sequence.As a result, be provided at represented nucleotide sequence in the Nucleotide 526 to 1048 of nucleotide sequence shown in the SEQ ID NO:227.

In addition, the chromosomal DNA that in embodiment 52 (1), prepares with restriction enzyme HincII digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:278 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:279 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1 to 600 of nucleotide sequence shown in the SEQ ID NO:237.

In addition, the chromosomal DNA that in embodiment 52 (1), prepares with restriction enzyme BalI digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:163 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:164 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 983 to 1449 of nucleotide sequence shown in the SEQ ID NO:237.

(3) sequential analysis of DNA of the present invention (A18)

By the nucleotide sequence that the nucleotide sequence acquisition that provided by embodiment 52 (2) DNA that obtain is represented in SEQ IDNO:237 is provided.In described nucleotide sequence, there are two open reading-frame (ORF)s (ORF).Therefore, comprise by 1230 Nucleotide (comprising terminator codon) and form and the nucleotide sequence (SEQ ID NO:227) of 409 amino-acid residues of encoding (SEQ ID NO:217) and form and the nucleotide sequence (SEQ ID NO:257) of 68 amino-acid residues (the SEQ IDNO:247) that encode by 207 Nucleotide (comprising terminator codon).The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:227 (SEQ ID NO:217) it is calculated that and is 45099Da.The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:257 (SEQ ID NO:247) it is calculated that and is 7193Da.

Embodiment 53 expression of DNA of the present invention (A18) in intestinal bacteria

(1) has the generation of the transformed into escherichia coli of DNA of the present invention (A18)

Except use from embodiment 52 (1) the chromosomal DNA that does not produce look streptomycete IFO 12735 preparations as template and use oligonucleotide with nucleotide sequence shown in the SEQ ID NO:183 and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:280 as the primer, be similar to embodiment 49 (1) and carry out PCR.Be similar to embodiment 32 (1), purify DNA and being cloned among the cloning vector pCRJI-TOPO (Invitrogen Company) from the PCR reaction soln.By having SEQ ID NO:67 respectively, the oligonucleotide of nucleotide sequence shown in 68,163,279 and 281 is as the nucleotide sequence of the plasmid DNA of primer analysis acquisition.Based on the result who obtains, the plasmid that will have nucleotide sequence shown in the SEQ ID NO:237 is called pCR646BF.Be similar to embodiment 32 (1), with restriction enzyme NdeI and HindIII digestion pCR646BF.The DNA of the about 1.5kbp of purifying from digestion product.Connect with the DNA of the acquisition of NdeI and HindIII digestion and plasmid pKSN2 to obtain to comprise the plasmid of nucleotide sequence shown in the SEQ ID NO:237, wherein with between the NdeI site and HindIII site of the DNA insertion pKSN2 of code book invention protein (A18) (hereinafter referred to as " pKSN646BF ").Described plasmid is imported e. coli jm109.The intestinal bacteria transformant that obtains is called JM109/pKSN646BF.

(2) expression and the described recovery of protein of protein of the present invention (A18) in intestinal bacteria

Be similar to embodiment 4 (2), cultivate among e. coli jm109/pKSN646BF and the JM109/pKSN2 each.Reclaim cell.The preparation cell pyrolysis liquid.Under embodiment 4 (2) described methods, preparation supernatant liquor fraction from cell pyrolysis liquid (below, to be called " intestinal bacteria pKSN646BF extract " available from the supernatant liquor fraction of e. coli jm109/pKSN646BF, will be called " intestinal bacteria pKSN2 extract ") available from the supernatant liquor fraction of e. coli jm109/pKSN2.

Be similar to embodiment 32 (3), prepare the reaction soln of 30 μ l and remain on 30 ℃ following 10 minutes.Yet,, use the supernatant liquor fraction (intestinal bacteria pKSN646BF extract or intestinal bacteria pKSN2 extract) of preparation in embodiment 53 (2) as the supernatant liquor fraction.Analyze with the reaction soln after the ethyl acetate extraction maintenance and with extract layer TLC.After launching the TLC plate, check on it corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).From the reaction soln that comprises intestinal bacteria pKSN646BF extract, detect spot corresponding to compound (III).Contrast does not detect this spot in the reaction soln that comprises intestinal bacteria pKSN2 extract therewith.

Embodiment 54 obtains DNA of the present invention (A19)

(1) preparation of the chromosomal DNA of streptomyces griseus IFO 13849T

According to embodiment 31 (1) described methods, the chromosomal DNA of preparation streptomyces griseus IFO 13849T.

(2) has the separation of the DNA of DNA of the present invention (A19) partial nucleotide sequence

According to embodiment 29 described methods, carry out PCR as template with by the use primer to 14 by the streptomyces griseus IFO13849T chromosomal DNA that will in embodiment 54 (1), prepare.Be similar to embodiment 31 (2), with the amplification dna clone to cloning vector pCRII-TOPO (InvitrogenCompany).Analyze its nucleotide sequence.As a result, be provided at represented nucleotide sequence in the Nucleotide 343 to 1069 of nucleotide sequence shown in the SEQ ID NO:228.

In addition, the chromosomal DNA that in embodiment 54 (1), prepares with restriction enzyme SmaI digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:282 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:283 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1 to 358 of nucleotide sequence shown in the SEQ ID NO:238.

In addition, the chromosomal DNA that in embodiment 54 (1), prepares with restriction enzyme HincII digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:284 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:285 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1005 to 1454 of nucleotide sequence shown in the SEQ ID NO:238.

(3) sequential analysis of DNA of the present invention (A19)

By the nucleotide sequence that the nucleotide sequence acquisition that provided by embodiment 54 (2) DNA that obtain is represented in SEQ IDNO:238 is provided.In described nucleotide sequence, there are two open reading-frame (ORF)s (ORF).Therefore, comprise by 1251 Nucleotide (comprising terminator codon) and form and the nucleotide sequence (SEQ ID NO:228) of 416 amino-acid residues of encoding (SEQ ID NO:218) and form and the nucleotide sequence (SEQ ID NO:258) of 51 amino-acid residues (the SEQ IDNO:248) that encode by 156 Nucleotide (comprising terminator codon).The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:228 (SEQ ID NO:218) it is calculated that and is 45903Da.The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:258 (SEQ ID NO:248) it is calculated that and is 5175Da.

Embodiment 55 expression of DNA of the present invention (A19) in intestinal bacteria

(1) has the generation of the transformed into escherichia coli of DNA of the present invention (A19)

Except the chromosomal DNA that uses streptomyces griseus IFO 13849T preparation from embodiment 54 (1) as template with use the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:286 and oligonucleotide as the primer with nucleotide sequence shown in the SEQ ID NO:287, be similar to embodiment 49 (1) and carry out PCR.Be similar to embodiment 32 (1), purify DNA and being cloned among the cloning vector pCRII-TOPO (Invitrogen Company) from the PCR reaction soln.By having SEQ ID NO:57 respectively, the oligonucleotide of nucleotide sequence shown in 59,284,286 and 288 is as the nucleotide sequence of the plasmid DNA of primer analysis acquisition.Based on the result who obtains, the plasmid that will have nucleotide sequence shown in the SEQ ID NO:238 is called pCR1502F.Be similar to embodiment 32 (1), with restriction enzyme NdeI and HindIII digestion pCR1502F.The DNA of the about 1.5kbp of purifying from digestion product.Connect with the DNA of the acquisition of NdeI and HindIII digestion and plasmid pKSN2 to obtain to comprise the plasmid of nucleotide sequence shown in the SEQ ID NO:238, wherein with between the NdeI site and HindIII site of the DNA insertion pKSN2 of code book invention protein (A19) (hereinafter referred to as " pKSN1502F ").Described plasmid is imported e. coli jm109.The intestinal bacteria transformant that obtains is called JM109/pKSN1502F.

(2) expression and the described recovery of protein of protein of the present invention (A19) in intestinal bacteria

Be similar to embodiment 4 (2), cultivate among e. coli jm109/pKSN1502F and the JM109/pKSN2 each.Reclaim cell.The preparation cell pyrolysis liquid.Under embodiment 4 (2) described methods, preparation supernatant liquor fraction from cell pyrolysis liquid (below, to be called " intestinal bacteria pKSN1502F extract " available from the supernatant liquor fraction of e. coli jm109/pKSN1502F, will be called " intestinal bacteria pKSN2 extract ") available from the supernatant liquor fraction of e. coli jm109/pKSN2.

Be similar to embodiment 32 (3), prepare the reaction soln of 30 μ 1 and remain on 30 ℃ following 10 minutes.Yet,, use the supernatant liquor fraction (intestinal bacteria pKSN1502F extract or intestinal bacteria pKSN2 extract) of preparation in embodiment 55 (2) as the supernatant liquor fraction.Analyze with the reaction soln after the ethyl acetate extraction maintenance and with extract layer TLC.After launching the TLC plate, check on it corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).From the reaction soln that comprises intestinal bacteria pKSN1502F extract, detect spot corresponding to compound (III).Contrast does not detect this spot in the reaction soln that comprises intestinal bacteria pKSN2 extract therewith.

Embodiment 56 obtains DNA of the present invention (A20)

(1) preparation of the chromosomal DNA of wool streptomycete IFO 12787T

According to embodiment 31 (1) described methods, the chromosomal DNA of preparation wool streptomycete IFO 12787T.

(2) has the separation of the DNA of DNA of the present invention (A20) partial nucleotide sequence

According to embodiment 29 described methods, carry out PCR as template with by the use primer to 14 by the wool streptomycete IFO 12787T chromosomal DNA that will in embodiment 56 (1), prepare.Be similar to embodiment 31 (2), with the amplification dna clone to cloning vector pCRII-TOPO (InvitrogenCompany).Analyze its nucleotide sequence.As a result, be provided at represented nucleotide sequence in the Nucleotide 304 to 1036 of nucleotide sequence shown in the SEQ ID NO:229.

In addition, the chromosomal DNA that in embodiment 56 (1), prepares with restriction enzyme PmacI digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:278 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:289 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1 to 318 of nucleotide sequence shown in the SEQ ID NO:239.

In addition, the chromosomal DNA that in embodiment 56 (1), prepares with restriction enzyme StuI digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:290 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:291 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 969 to 1461 of nucleotide sequence shown in the SEQ ID NO:239.

(3) sequential analysis of DNA of the present invention (A20)

By the nucleotide sequence that the nucleotide sequence acquisition that provided by embodiment 56 (2) DNA that obtain is represented in SEQ IDNO:239 is provided.In described nucleotide sequence, there are two open reading-frame (ORF)s (ORF).Therefore, comprise by 1218 Nucleotide (comprising terminator codon) and form and the nucleotide sequence (SEQ ID NO:229) of 405 amino-acid residues of encoding (SEQ ID NO:219) and form and the nucleotide sequence (SEQ ID NO:259) of 76 amino-acid residues (the SEQ IDNO:249) that encode by 231 Nucleotide (comprising terminator codon).The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:229 (SEQ ID NO:219) it is calculated that and is 45071Da.The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:259 (SEQ ID NO:249) it is calculated that and is 7816Da.

Embodiment 57 expression of DNA of the present invention (A20) in intestinal bacteria

(1) has the generation of the transformed into escherichia coli of DNA of the present invention (A20)

Except use from the chromosomal DNA of embodiment 56 (1) wool streptomycete IFO 12787T preparation as template and use oligonucleotide with nucleotide sequence shown in the SEQ ID NO:292 and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:293 as the primer, be similar to embodiment 49 (1) and carry out PCR.Be similar to embodiment 32 (1), purify DNA and being cloned among the cloning vector pCRII-TOPO (Invitrogen Company) from the PCR reaction soln.By having SEQ ID NO:67 respectively, the oligonucleotide of nucleotide sequence shown in 68,188,278 and 290 is as the nucleotide sequence of the plasmid DNA of primer analysis acquisition.Based on the result who obtains, the plasmid that will have nucleotide sequence shown in the SEQ IDNO:239 is called pCR1525F.Be similar to embodiment 32 (1), with restriction enzyme NdeI and HindIII digestion pCR1525F.The DNA of the about 1.5kbp of purifying from digestion product.Connect with the DNA of the acquisition of NdeI and HindIII digestion and plasmid pKSN2 to obtain to comprise the plasmid of nucleotide sequence shown in the SEQ ID NO:239, wherein with between the NdeI site and HindIII site of the DNA insertion pKSN2 of code book invention protein (A20) (hereinafter referred to as " pKSN1525F ").Described plasmid is imported e. coli jm109.The intestinal bacteria transformant that obtains is called JM109/pKSN1525F.

(2) expression and the described recovery of protein of protein of the present invention (A20) in intestinal bacteria

Be similar to embodiment 4 (2), cultivate among e. coli jm109/pKSN1525F and the JM109/pKSN2 each.Reclaim cell.The preparation cell pyrolysis liquid.Under embodiment 4 (2) described methods, preparation supernatant liquor fraction from cell pyrolysis liquid (below, to be called " intestinal bacteria pKSN1525F extract " available from the supernatant liquor fraction of e. coli jm109/pKSN1525F, will be called " intestinal bacteria pKSN2 extract ") available from the supernatant liquor fraction of e. coli jm109/pKSN2.

Be similar to embodiment 32 (3), prepare the reaction soln of 30 μ l and remain on 30 ℃ following 10 minutes.Yet,, use the supernatant liquor fraction (intestinal bacteria pKSN1525F extract or intestinal bacteria pKSN2 extract) of preparation in embodiment 57 (2) as the supernatant liquor fraction.Analyze with the reaction soln after the ethyl acetate extraction maintenance and with extract layer TLC.After launching the TLC plate, check on it corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).From the reaction soln that comprises intestinal bacteria pKSN1525F extract, detect spot corresponding to compound (III).Contrast does not detect this spot in the reaction soln that comprises intestinal bacteria pKSN2 extract therewith.

Embodiment 58 obtains DNA of the present invention (A21)

The preparation of the chromosomal DNA of (1) three damp streptomycete IFO 13855T

According to embodiment 31 (1) described methods, prepare the chromosomal DNA of three damp streptomycete IFO 13855T.

(2) has the separation of the DNA of DNA of the present invention (A21) partial nucleotide sequence

According to embodiment 29 described methods, carry out PCR as template with by the use primer to 14 by the three damp streptomycete IFO13855T chromosomal DNAs that will in embodiment 58 (1), prepare.Be similar to embodiment 31 (2), with the amplification dna clone to cloning vector pCRII-TOPO (InvitrogenCompany).Analyze its nucleotide sequence.As a result, be provided at represented nucleotide sequence in the Nucleotide 328 to 1063 of nucleotide sequence shown in the SEQ ID NO:230.

In addition, the chromosomal DNA that in embodiment 58 (1), prepares with restriction enzyme SmaI digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:294 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:295 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1 to 341 of nucleotide sequence shown in the SEQ ID NO:240.

In addition, the chromosomal DNA that in embodiment 58 (1), prepares with restriction enzyme HincII digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:296 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:297 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1017 to 1458 of nucleotide sequence shown in the SEQ ID NO:240.

(3) sequential analysis of DNA of the present invention (A21)

By the nucleotide sequence that the nucleotide sequence acquisition that provided by embodiment 58 (2) DNA that obtain is represented in SEQ IDNO:240 is provided.In described nucleotide sequence, there are two open reading-frame (ORF)s (ORF).Therefore, comprise by 1245 Nucleotide (comprising terminator codon) and form and the nucleotide sequence (SEQ ID NO:230) of 414 amino-acid residues of encoding (SEQ ID NO:220) and form and the nucleotide sequence (SEQ ID NO:260) of 66 amino-acid residues (the SEQ IDNO:250) that encode by 201 Nucleotide (comprising terminator codon).The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:230 (SEQ ID NO:220) it is calculated that and is 45806Da.The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:260 (SEQ ID NO:250) it is calculated that and is 6712Da.

Embodiment 59 expression of DNA of the present invention (A21) in intestinal bacteria

(1) has the generation of the transformed into escherichia coli of DNA of the present invention (A21)

Except the chromosomal DNA that uses three damp streptomycete IFO 13855T preparations from embodiment 58 (1) as template and use oligonucleotide with nucleotide sequence shown in the SEQ ID NO:298 and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:299 as the primer, be similar to embodiment 32 (1) and carry out PCR.Be similar to embodiment 32 (1), purify DNA and being cloned among the cloning vector pCRII-TOPO (Invitrogen Company) from the PCR reaction soln.By having SEQ ID NO:57 respectively, the oligonucleotide of nucleotide sequence shown in 59,296 and 300 is as the nucleotide sequence of the plasmid DNA of primer analysis acquisition.Based on the result who obtains, the plasmid that will have nucleotide sequence shown in the SEQ ID NO:240 is called pCR1543BF.Be similar to embodiment 32 (1), with restriction enzyme NdeI and HindIII digestion pCR1543BF.The DNA of the about 1.5kbp of purifying from digestion product.Connect with the DNA of the acquisition of NdeI and HindIII digestion and plasmid pKSN2 to obtain to comprise the plasmid of nucleotide sequence shown in the SEQ ID NO:240, wherein with between the NdeI site and HindIII site of the DNA insertion pKSN2 of code book invention protein (A21) (hereinafter referred to as " pKSN1543BF ").Described plasmid is imported e. coli jm109.The intestinal bacteria transformant that obtains is called JM109/pKSN1543BF.

(2) expression and the described recovery of protein of protein of the present invention (A21) in intestinal bacteria

Be similar to embodiment 4 (2), cultivate among e. coli jm109/pKSN1543BF and the JM109/pKSN2 each.Reclaim cell.The preparation cell pyrolysis liquid.Under embodiment 4 (2) described methods, preparation supernatant liquor fraction from cell pyrolysis liquid (below, to be called " intestinal bacteria pKSN1543BF extract " available from the supernatant liquor fraction of e. coli jm109/pKSN1543BF, will be called " intestinal bacteria pKSN2 extract ") available from the supernatant liquor fraction of e. coli jm109/pKSN2.

Be similar to embodiment 32 (3), prepare the reaction soln of 30 μ l and remain on 30 ℃ following 10 minutes.Yet,, use the supernatant liquor fraction (intestinal bacteria pKSN1543BF extract or intestinal bacteria pKSN2 extract) of preparation in embodiment 59 (2) as the supernatant liquor fraction.Analyze with the reaction soln after the ethyl acetate extraction maintenance and with extract layer TLC.After launching the TLC plate, check on it corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).From the reaction soln that comprises intestinal bacteria pKSN1543BF extract, detect spot corresponding to compound (III).Contrast does not detect this spot in the reaction soln that comprises intestinal bacteria pKSN2 extract therewith.

Embodiment 60 obtains DNA of the present invention (A22)

(1) preparation of the chromosomal DNA of pale asphyxia streptomycete IFO13434T

According to embodiment 31 (1) described methods, the chromosomal DNA of preparation pale asphyxia streptomycete IFO 13434T.

(2) has the separation of the DNA of DNA of the present invention (A22) partial nucleotide sequence

According to embodiment 29 described methods, carry out PCR as template with by the use primer to 15 by the pale asphyxia streptomycete IFO1 3434T chromosomal DNA that will in embodiment 60 (1), prepare.Be similar to embodiment 31 (2), with the amplification dna clone to cloning vector pCRII-TOPO (Invitrogen Company).Analyze its nucleotide sequence.As a result, be provided at represented nucleotide sequence in the Nucleotide 483 to 1048 of nucleotide sequence shown in the SEQ ID NO:231.

In addition, the chromosomal DNA that in embodiment 60 (1), prepares with restriction enzyme SmaI digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:301 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:302 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 68 to 516 of nucleotide sequence shown in the SEQ ID NO:241.

In addition, the chromosomal DNA that in embodiment 60 (1), prepares with restriction enzyme HincII digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:302 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:303 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1 to 270 of nucleotide sequence shown in the SEQ ID NO:241.

In addition, the chromosomal DNA that in embodiment 60 (1), prepares with restriction enzyme HincII digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:304 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:305 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 982 to 1448 of nucleotide sequence shown in the SEQ ID NO:241.

(3) sequential analysis of DNA of the present invention (A22)

By the nucleotide sequence that the nucleotide sequence acquisition that provided by embodiment 60 (2) DNA that obtain is represented in SEQ IDNO:241 is provided.In described nucleotide sequence, there are two open reading-frame (ORF)s (ORF).Therefore, comprise by 1230 Nucleotide (comprising terminator codon) and form and the nucleotide sequence (SEQ ID NO:231) of 409 amino-acid residues of encoding (SEQ ID NO:221) and form and the nucleotide sequence (SEQ ID NO:261) of 64 amino-acid residues (the SEQ IDNO:251) that encode by 195 Nucleotide (comprising terminator codon).The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:231 (SEQ ID NO:221) it is calculated that and is 45050Da.The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:261 (SEQ ID NO:251) it is calculated that and is 6914Da.

Embodiment 61 expression of DNA of the present invention (A22) in intestinal bacteria

(1) has the generation of the transformed into escherichia coli of DNA of the present invention (A22)

Except the chromosomal DNA that uses pale asphyxia streptomycete IFO 13434T preparation from embodiment 60 (1) as template with use the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:306 and oligonucleotide as the primer with nucleotide sequence shown in the SEQ ID NO:307, be similar to embodiment 32 (1) and carry out PCR.Be similar to embodiment 32 (1), purify DNA and being cloned among the cloning vector pCRII-TOPO (Invitrogen Company) from the PCR reaction soln.By having SEQ ID NO:67 respectively, the oligonucleotide of nucleotide sequence shown in 68 and 308 is as the nucleotide sequence of the plasmid DNA of primer analysis acquisition.Based on the result who obtains, the plasmid that will have nucleotide sequence shown in the SEQ IDNO:241 is called pCR1558BF.Be similar to embodiment 32 (1), with restriction enzyme NdeI and HindIII digestion pCR1558BF.The DNA of the about 1.5kbp of purifying from digestion product.Connect with the DNA of the acquisition of NdeI and HindIII digestion and plasmid pKSN2 to obtain to comprise the plasmid of nucleotide sequence shown in the SEQ ID NO:241, wherein with between the NdeI site and HindIII site of the DNA insertion pKSN2 of code book invention protein (A22) (hereinafter referred to as " pKSN1558BF ").Described plasmid is imported e. coli jm109.The intestinal bacteria transformant that obtains is called JM109/pKSN1558BF.

(2) expression and the described recovery of protein of protein of the present invention (A22) in intestinal bacteria

Be similar to embodiment 4 (2), cultivate among e. coli jm109/pKSN1558BF and the JM109/pKSN2 each.Reclaim cell.The preparation cell pyrolysis liquid.Under embodiment 4 (2) described methods, preparation supernatant liquor fraction from cell pyrolysis liquid (below, to be called " intestinal bacteria pKSN1558BF extract " available from the supernatant liquor fraction of e. coli jm109/pKSN1558BF, will be called " intestinal bacteria pKSN2 extract ") available from the supernatant liquor fraction of e. coli jm109/pKSN2.

Be similar to embodiment 32 (3), prepare the reaction soln of 30 μ l and remain on 30 ℃ following 10 minutes.Yet,, use the supernatant liquor fraction (intestinal bacteria pKSN1558BF extract or intestinal bacteria pKSN2 extract) of preparation in embodiment 61 (2) as the supernatant liquor fraction.Analyze with the reaction soln after the ethyl acetate extraction maintenance and with extract layer TLC.After launching the TLC plate, check on it corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).From the reaction soln that comprises intestinal bacteria pKSN1558BF extract, detect spot corresponding to compound (III).Contrast does not detect this spot in the reaction soln that comprises intestinal bacteria pKSN2 extract therewith.

Embodiment 62 obtains DNA of the present invention (A23)

(1) the rose preparation of chromosomal DNA of streptomycete IFO 13682T that reddens

According to embodiment 31 (1) described methods, the redden chromosomal DNA of streptomycete IFO 13682T of preparation rose.

(2) has the separation of the DNA of DNA of the present invention (A23) partial nucleotide sequence

According to embodiment 29 described methods, redden streptomycete IFO13682T chromosomal DNA as template with by using primer to carry out PCR to 14 by the rose that will in embodiment 62 (1), prepare.Be similar to embodiment 31 (2), with the amplification dna clone to cloning vector pCRII-TOPO (Invitrogen Company).Analyze its nucleotide sequence.As a result, be provided at represented nucleotide sequence in the Nucleotide 289 to 1015 of nucleotide sequence shown in the SEQ ID NO:232.

In addition, the chromosomal DNA that in embodiment 62 (1), prepares with restriction enzyme SmaI digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:309 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:310 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1 to 354 of nucleotide sequence shown in the SEQ ID NO:242.

In addition, the chromosomal DNA that in embodiment 62 (1), prepares with restriction enzyme PvuII digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:311 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:312 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 966 to 1411 of nucleotide sequence shown in the SEQ ID NO:242.

(3) sequential analysis of DNA of the present invention (A23)

By the nucleotide sequence that the nucleotide sequence acquisition that provided by embodiment 62 (2) DNA that obtain is represented in SEQ IDNO:242 is provided.In described nucleotide sequence, there are two open reading-frame (ORF)s (ORF).Therefore, comprise by 1197 Nucleotide (comprising terminator codon) and form and the nucleotide sequence (SEQ ID NO:232) of 398 amino-acid residues of encoding (SEQ ID NO:222) and form and the nucleotide sequence (SEQ ID NO:262) of 66 amino-acid residues (the SEQ IDNO:252) that encode by 201 Nucleotide (comprising terminator codon).The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:232 (SEQ ID NO:222) it is calculated that and is 43624Da.The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:262 (SEQ ID NO:252) it is calculated that and is 6797Da.

Embodiment 63 expression of DNA of the present invention (A23) in intestinal bacteria

(1) has the generation of the transformed into escherichia coli of DNA of the present invention (A23)

Except use among embodiment 62 (1) rose redden the chromosomal DNA of streptomycete IFO13682T preparation as template and use oligonucleotide with nucleotide sequence shown in the SEQ ID NO:313 and oligonucleotide with nucleotide sequence shown in the SEQ ID NO:314 as primer, be similar to embodiment 49 (1) and carry out PCR.Be similar to embodiment 32 (1), purify DNA and being cloned among the cloning vector pCRII-TOPO (Invitrogen Company) from the PCR reaction soln.By having SEQ ID NO:67 respectively, the oligonucleotide of nucleotide sequence shown in 68,309,311 and 315 is as the nucleotide sequence of the plasmid DNA of primer analysis acquisition.Based on the result who obtains, the plasmid that will have nucleotide sequence shown in the SEQ ID NO:242 is called pCR1584F.Be similar to embodiment 32 (1), with restriction enzyme NdeI and HindIII digestion pCR1584F.The DNA of the about 1.5kbp of purifying from digestion product.Connect with the DNA of the acquisition of NdeI and HindIII digestion and plasmid pKSN2 to obtain to comprise the plasmid of nucleotide sequence shown in the SEQ ID NO:242, wherein with between the NdeI site and HindIII site of the DNA insertion pKSN2 of code book invention protein (A23) (hereinafter referred to as " pKSN1584F ").Described plasmid is imported e. coli jm109.The intestinal bacteria transformant that obtains is called JM109/pKSN1584F.

(2) expression and the described recovery of protein of protein of the present invention (A23) in intestinal bacteria

Be similar to embodiment 4 (2), cultivate among e. coli jm109/pKSN1584F and the JM109/pKSN2 each.Reclaim cell.The preparation cell pyrolysis liquid.Under embodiment 4 (2) described methods, preparation supernatant liquor fraction from cell pyrolysis liquid (below, to be called " intestinal bacteria pKSN1584F extract " available from the supernatant liquor fraction of e. coli jm109/pKSN1584F, will be called " intestinal bacteria pKSN2 extract ") available from the supernatant liquor fraction of e. coli jm109/pKSN2.

Be similar to embodiment 32 (3), prepare the reaction soln of 30 μ l and remain on 30 ℃ following 10 minutes.Yet,, use the supernatant liquor fraction (intestinal bacteria pKSN1584F extract or intestinal bacteria pKSN2 extract) of preparation in embodiment 63 (2) as the supernatant liquor fraction.Analyze with the reaction soln after the ethyl acetate extraction maintenance and with extract layer TLC.After launching the TLC plate, check on it corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).From the reaction soln that comprises intestinal bacteria pKSN1584F extract, detect spot corresponding to compound (III).Contrast does not detect this spot in the reaction soln that comprises intestinal bacteria pKSN2 extract therewith.

Embodiment 64 obtains DNA of the present invention (A24)

(1) preparation of the chromosomal DNA of ground, Shandong streptomycete IFO 15875T

According to embodiment 31 (1) described methods, the chromosomal DNA of ground, preparation Shandong streptomycete IFO 15875T.

(2) has the separation of the DNA of DNA of the present invention (A24) partial nucleotide sequence

According to embodiment 29 described methods, by will in the embodiment 64 (1) of preparation, carrying out PCR as template with by the use primer to 14 by ground, Shandong streptomycete IFO15875T chromosomal DNA.Be similar to embodiment 31 (2), with the amplification dna clone to cloning vector pCRII-TOPO (InvitrogenCompany).Analyze its nucleotide sequence.As a result, be provided at represented nucleotide sequence in the Nucleotide 322 to 1057 of nucleotide sequence shown in the SEQ ID NO:233.

In addition, the chromosomal DNA that in embodiment 64 (1), prepares with restriction enzyme SmaI digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:316 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:317 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1 to 384 of nucleotide sequence shown in the SEQ ID NO:243.

In addition, the chromosomal DNA that in embodiment 64 (1), prepares with restriction enzyme NaeI digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:318 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:319 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 992 to 1466 of nucleotide sequence shown in the SEQ ID NO:243.

(3) sequential analysis of DNA of the present invention (A24)

By the nucleotide sequence that the nucleotide sequence acquisition that provided by embodiment 64 (2) DNA that obtain is represented in SEQ IDNO:243 is provided.In described nucleotide sequence, there are two open reading-frame (ORF)s (ORF).Therefore, comprise by 1245 Nucleotide (comprising terminator codon) and form and the nucleotide sequence (SEQ ID NO:233) of 414 amino-acid residues of encoding (SEQ ID NO:223) and form and the nucleotide sequence (SEQ ID NO:263) of 65 amino-acid residues (the SEQ IDNO:253) that encode by 198 Nucleotide (comprising terminator codon).The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:233 (SEQ ID NO:223) it is calculated that and is 45830Da.The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:263 (SEQ ID NO:253) it is calculated that and is 7034Da.

Embodiment 65 expression of DNA of the present invention (A24) in intestinal bacteria

(1) has the generation of the transformed into escherichia coli of DNA of the present invention (A24)

Except the chromosomal DNA that uses ground, Shandong streptomycete IFO15875T preparation among embodiment 64 (1) as template and use oligonucleotide with nucleotide sequence shown in the SEQ ID NO:320 and oligonucleotide with nucleotide sequence shown in the SEQ ID NO:321 as primer, be similar to embodiment 49 (1) and carry out PCR.Be similar to embodiment 32 (1), purify DNA and being cloned among the cloning vector pCRII-TOPO (Invitrogen Company) from the PCR reaction soln.By having SEQ ID NO:67 respectively, the oligonucleotide of nucleotide sequence shown in 68 and 322 is as the nucleotide sequence of the plasmid DNA of primer order-checking acquisition.Based on the result who obtains, the plasmid that will have nucleotide sequence shown in the SEQ IDNO:243 is called pCR1589BF.Be similar to embodiment 32 (1), with restriction enzyme NdeI and HindIII digestion pCR1589BF.The DNA of the about 1.5kbp of purifying from digestion product.Connect with the DNA of the acquisition of NdeI and HindIII digestion and plasmid pKSN2 to obtain to comprise the plasmid of nucleotide sequence shown in the SEQ ID NO:243, wherein with between the NdeI site and HindIII site of the DNA insertion pKSN2 of code book invention protein (A24) (hereinafter referred to as " pKSN1589BF ").Described plasmid is imported e. coli jm109.The intestinal bacteria transformant that obtains is called JM109/pKSN1589BF.

(2) expression and the described recovery of protein of protein of the present invention (A24) in intestinal bacteria

Be similar to embodiment 4 (2), cultivate among e. coli jm109/pKSN1589BF and the JM109/pKSN2 each.Reclaim cell.The preparation cell pyrolysis liquid.Under embodiment 4 (2) described methods, preparation supernatant liquor fraction from cell pyrolysis liquid (below, to be called " intestinal bacteria pKSN1589BF extract " available from the supernatant liquor fraction of e. coli jm109/pKSN1589BF, will be called " intestinal bacteria pKSN2 extract ") available from the supernatant liquor fraction of e. coli jm109/pKSN2.

Be similar to embodiment 32 (3), prepare the reaction soln of 30 μ l and remain on 30 ℃ following 10 minutes.Yet,, use the supernatant liquor fraction (intestinal bacteria pKSN1589BF extract or intestinal bacteria pKSN2 extract) of preparation in embodiment 65 (2) as the supernatant liquor fraction.Analyze with the reaction soln after the ethyl acetate extraction maintenance and with extract layer TLC.After launching the TLC plate, check on it corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).From the reaction soln that comprises intestinal bacteria pKSN1589BF extract, detect spot corresponding to compound (III).Contrast does not detect this spot in the reaction soln that comprises intestinal bacteria pKSN2 extract therewith.

Embodiment 66 obtains DNA of the present invention (A25)

(1) preparation of the chromosomal DNA of Regensburg streptomycete IFO 13446T

According to embodiment 31 (1) described methods, the chromosomal DNA of preparation Regensburg streptomycete IFO 13446T.

(2) has the separation of the DNA of DNA of the present invention (A25) partial nucleotide sequence

According to embodiment 29 described methods, carry out PCR as template with by the use primer to 14 by the Regensburg streptomycete IFO13446T chromosomal DNA that will in embodiment 66 (1), prepare.Be similar to embodiment 31 (2), with the amplification dna clone to cloning vector pCRII-TOPO (InvitrogenCompany).Analyze its nucleotide sequence.As a result, be provided at represented nucleotide sequence in the Nucleotide 289 to 1015 of nucleotide sequence shown in the SEQ ID NO:234.

In addition, the chromosomal DNA that in embodiment 66 (1), prepares with restriction enzyme SmaI digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:323 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:324 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 1 to 303 of nucleotide sequence shown in the SEQ ID NO:244.

In addition, the chromosomal DNA that in embodiment 66 (1), prepares with restriction enzyme PmacI digestion.According to embodiment 26 (3) described methods, produce genome walking library by using the DNA that obtains.Have the oligonucleotide and the primer AP1 of nucleotide sequence shown in the SEQ ID NO:311 by the library that use to obtain as template with by use, under embodiment 26 (3) described conditions, carry out PCR to obtain a PCR product.Next, have the oligonucleotide and the primer AP2 of nucleotide sequence shown in the SEQ ID NO:325 as template with by use, under embodiment 26 (3) described conditions, carry out PCR by using a PCR product.Analyze the nucleotide sequence of the DNA that obtains.Be provided at represented nucleotide sequence in the Nucleotide 966 to 1411 of nucleotide sequence shown in the SEQ ID NO:244.

(3) sequential analysis of DNA of the present invention (A25)

By the nucleotide sequence that the nucleotide sequence acquisition that provided by embodiment 66 (2) DNA that obtain is represented in SEQ IDNO:244 is provided.In described nucleotide sequence, there are two open reading-frame (ORF)s (ORF).Therefore, comprise by the former times acid of 1197 nuclear and (comprising terminator codon) form and the nucleotide sequence (SEQ ID NO:234) of 398 amino-acid residues of encoding (SEQ ID NO:224) and form and the nucleotide sequence (SEQ ID NO:264) of 66 amino-acid residues (the SEQ IDNO:254) that encode by 201 Nucleotide (comprising terminator codon).The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:234 (SEQ ID NO:224) it is calculated that and is 44175Da.The proteinic molecular weight of being made up of aminoacid sequence nucleotide sequence coded shown in the SEQ ID NO:264 (SEQ ID NO:254) it is calculated that and is 6685Da.

Embodiment 67 expression of DNA of the present invention (A25) in intestinal bacteria

(1) has the generation of the transformed into escherichia coli of DNA of the present invention (A25)

Except the chromosomal DNA that uses Regensburg streptomycete IFO13446T preparation among embodiment 66 (1) as template and use oligonucleotide with nucleotide sequence shown in the SEQ ID NO:326 and oligonucleotide with nucleotide sequence shown in the SEQ ID NO:327 as primer, be similar to embodiment 49 (1) and carry out PCR.Be similar to embodiment 32 (1), purify DNA and being cloned among the cloning vector pCRII-TOPO (Invitrogen Company) from the PCR reaction soln.By having SEQ ID NO:67 respectively, the oligonucleotide of nucleotide sequence shown in 68,311,315 and 323 is as the nucleotide sequence of the plasmid DNA of primer order-checking acquisition.Based on the result who obtains, the plasmid that will have nucleotide sequence shown in the SEQ ID NO:244 is called pCR1609F.Be similar to embodiment 32 (1), with restriction enzyme NdeI and HindIII digestion pCR1609F.The DNA of the about 1.5kbp of purifying from digestion product.Connect with the DNA of the acquisition of NdeI and HindIII digestion and plasmid pKSN2 to obtain to comprise the plasmid of nucleotide sequence shown in the SEQ ID NO:244, wherein with between the NdeI site and HindIII site of the DNA insertion pKSN2 of code book invention protein (A25) (hereinafter referred to as " pKSN1609F ").Described plasmid is imported e. coli jm109.The intestinal bacteria transformant that obtains is called JM109/pKSN1609F.

(2) expression and the described recovery of protein of protein of the present invention (A25) in intestinal bacteria

Be similar to embodiment 4 (2), cultivate among e. coli jm109/pKSN1609F and the JM109/pKSN2 each.Reclaim cell.The preparation cell pyrolysis liquid.Under embodiment 4 (2) described methods, preparation supernatant liquor fraction from cell pyrolysis liquid (below, to be called " intestinal bacteria pKSN1609F extract " available from the supernatant liquor fraction of e. coli jm109/pKSN1609F, will be called " intestinal bacteria pKSN2 extract ") available from the supernatant liquor fraction of e. coli jm109/pKSN2.

Be similar to embodiment 32 (3), prepare the reaction soln of 30 μ l and remain on 30 ℃ following 10 minutes.Yet,, use the supernatant liquor fraction (intestinal bacteria pKSN1609F extract or intestinal bacteria pKSN2 extract) of preparation in embodiment 67 (2) as the supernatant liquor fraction.Analyze with the reaction soln after the ethyl acetate extraction maintenance and with extract layer TLC.After launching the TLC plate, check on it corresponding to usefulness ¹⁴The existing of the spot of the compound of C mark (III) (Rf value 0.24 and 0.29).From the reaction soln that comprises intestinal bacteria pKSN1609F extract, detect spot corresponding to compound (III).Contrast does not detect this spot in the reaction soln that comprises intestinal bacteria pKSN2 extract therewith.

Embodiment 68 is by protein of the present invention (A16), (A17), and (A18), (A19), (A20), (A21), (A22), (A23), (A24) or the metabolism of compound (A25)

(1) metabolism of the compound (XII) by protein of the present invention (A16)

100 μ l reaction solns of preparation 50mM potassiumphosphate (pH7.0), it comprises 12.5ppm compound (XII), 3mM p-NADPH (hereinafter referred to as " component A ") (Oriental YeastCompany), the ferredoxin (hereinafter referred to as " B component ") that 1mg/ml derives from spinach (SigmaCompany), 0.15U/ml ferredoxin reductase (hereinafter referred to as " component C ") is (SigmaCompany) and the supernatant liquor fraction that reclaims in embodiment 49 (2) of 20 μ l.With reaction soln remain on 30 ℃ following 10 minutes.In addition, prepare similarly and keep not to add be used for above-mentioned reaction soln composition, be selected from component A, 100 μ l reaction solns of the 50mM potassium phosphate buffer (pH 7.0) of at least a component of B component and component C and the supernatant liquor fraction that among embodiment 49 (2), prepares.Five microlitres (5 μ l) 2N HCl and 100 μ l ethyl acetate are added and be mixed in the every kind of reaction soln that keeps later.Filter 8 centrifugal supernatant liquor under the 000xg with UltraFree MC 0.22 μ m filter device (MilliporeCompany).Above-mentioned analysis condition 1 time analyzing on the HPLC 40 microlitres (40 μ l) liquid permeate (below, self-contained component A in the future, B component, the liquid permeate of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 49 (2) are called " (XII) metabolism solution (A16) "; In addition, will not comprise B component from not comprising component A, the liquid permeate that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 49 (2) is called " (XII) contrast solution (A16) ").Compare with the concentration of the compound (XII) that detects from (XII) contrast solution (A16), the concentration of the compound (XII) that detects from (XII) metabolism solution (A16) is lower.Detect the peak from (XII) metabolism solution (A16) in addition, it does not detect from (XII) contrast solution (A16).The peak elution time of the compound of the described compound (XII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (2) (XII) metabolism solution (A1) matches.

(2) metabolism of the compound (XII) by protein of the present invention (A17)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 49 (2) in embodiment 51 (2), according to embodiment 68 (1) described methods preparations with keep reaction solns.Be similar to embodiment 68 (1), the reaction soln after preparation keeps.Above-mentioned analysis condition 1 time analyzing on the HPLC liquid permeate that 40 microlitres (40 μ l) obtain (below, self-contained component A in the future, B component, the liquid permeate of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 51 (2) are called " (XII) metabolism solution (A17) "; In addition, will not comprise B component from not comprising component A, the liquid permeate that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 51 (2) is called " (XII) contrast solution (A17) ").Compare with the concentration of the compound (XII) that detects from (XII) contrast solution (A17), the concentration of the compound (XII) that detects from (XII) metabolism solution (A17) is lower.Detect the peak from (XII) metabolism solution (A17) in addition, it does not detect from (XII) contrast solution (A17).The peak elution time of the compound of the described compound (XII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (2) (XII) metabolism solution (A1) matches.

(3) metabolism of the compound (XII) by protein of the present invention (A18)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 49 (2) in embodiment 53 (2), according to embodiment 68 (1) described methods preparations with keep reaction solns.Be similar to embodiment 68 (1), every kind of reaction soln after preparation keeps.Above-mentioned analysis condition 1 time analyzing on the HPLC liquid permeate that 40 microlitres (40 μ l) obtain (below, self-contained component A in the future, B component, the liquid permeate of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 53 (2) are called " (XII) metabolism solution (A18) "; In addition, will not comprise B component from not comprising component A, the liquid permeate that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 53 (2) is called " (XII) contrast solution (A18) ").Compare with the concentration of the compound (XII) that detects from (XII) contrast solution (A18), the concentration of the compound (XII) that detects from (XII) metabolism solution (A18) is lower.Detect the peak from (XII) metabolism solution (A18) in addition, it does not detect from (XII) contrast solution (A18).The peak elution time of the compound of the described compound (XII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (2) (XII) metabolism solution (A1) matches.

(4) metabolism of the compound (XII) by protein of the present invention (A19)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 49 (2) in embodiment 55 (2), according to embodiment 68 (1) described methods preparations with keep reaction solns.Be similar to embodiment 68 (1), every kind of reaction soln after preparation keeps.Above-mentioned analysis condition 1 time analyzing on the HPLC liquid permeate that 40 microlitres (40 μ l) obtain (below, self-contained component A in the future, B component, the liquid permeate of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 55 (2) are called " (XII) metabolism solution (A19) "; In addition, will not comprise B component from not comprising component A, the liquid permeate that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 55 (2) is called " (XII) contrast solution (A19) ").Compare with the concentration of the compound (XII) that detects from (XII) contrast solution (A19), the concentration of the compound (XII) that detects from (XII) metabolism solution (A19) is lower.Detect the peak from (XII) metabolism solution (A19) in addition, it does not detect from (XII) contrast solution (A19).The peak elution time of the compound of the described compound (XII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (2) (XII) metabolism solution (A1) matches.

(5) metabolism of the compound (XII) by protein of the present invention (A20)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 49 (2) in embodiment 57 (2), according to embodiment 68 (1) described methods preparations with keep reaction solns.Be similar to embodiment 68 (1), every kind of reaction soln after preparation keeps.Above-mentioned analysis condition 1 time analyzing on the HPLC liquid permeate that 40 microlitres (40 μ l) obtain (below, self-contained component A in the future, B component, the liquid permeate of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 57 (2) are called " (XII) metabolism solution (A20) "; In addition, will not comprise B component from not comprising component A, the liquid permeate that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 57 (2) is called " (XII) contrast solution (A20) ").Compare with the concentration of the compound (XII) that detects from (XII) contrast solution (A20), the concentration of the compound (XII) that detects from (XII) metabolism solution (A20) is lower.Detect the peak from (XII) metabolism solution (A20) in addition, it does not detect from (XII) contrast solution (A20).The peak elution time of the compound of the described compound (XII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (2) (XII) metabolism solution (A1) matches.

(6) metabolism of the compound (XII) by protein of the present invention (A21)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 49 (2) in embodiment 59 (2), according to embodiment 68 (1) described methods preparations with keep reaction solns.Be similar to embodiment 68 (1), every kind of reaction soln after preparation keeps.Above-mentioned analysis condition 1 time analyzing on the HPLC liquid permeate that 40 microlitres (40 μ l) obtain (below, self-contained component A in the future, B component, the liquid permeate of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 59 (2) are called " (XII) metabolism solution (A21) "; In addition, will not comprise B component from not comprising component A, the liquid permeate that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 59 (2) is called " (XII) contrast solution (A21) ").Compare with the concentration of the compound (XII) that detects from (XII) contrast solution (A21), the concentration of the compound (XII) that detects from (XII) metabolism solution (A21) is lower.Detect the peak from (XII) metabolism solution (A21) in addition, it does not detect from (XII) contrast solution (A21).The peak elution time of the compound of the described compound (XII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (2) (XII) metabolism solution (A1) matches.

(7) metabolism of the compound (XII) by protein of the present invention (A22)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 49 (2) in embodiment 61 (2), according to embodiment 68 (1) described methods preparations with keep reaction solns.Be similar to embodiment 68 (1), every kind of reaction soln after preparation keeps.Above-mentioned analysis condition 1 time analyzing on the HPLC liquid permeate that 40 microlitres (40 μ l) obtain (below, self-contained component A in the future, B component, the liquid permeate of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 61 (2) are called " (XII) metabolism solution (A22) "; In addition, will not comprise B component from not comprising component A, the liquid permeate that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 61 (2) is called " (XII) contrast solution (A22) ").Compare with the concentration of the compound (XII) that detects from (XII) contrast solution (A22), the concentration of the compound (XII) that detects from (XII) metabolism solution (A22) is lower.Detect the peak from (XII) metabolism solution (A22) in addition, it does not detect from (XII) contrast solution (A22).The peak elution time of the compound of the described compound (XII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (2) (XII) metabolism solution (A1) matches.

(8) metabolism of the compound (XII) by protein of the present invention (A23)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 49 (2) in embodiment 63 (2), according to embodiment 68 (1) described methods preparations with keep reaction solns.Be similar to embodiment 68 (1), every kind of reaction soln after preparation keeps.Above-mentioned analysis condition 1 time analyzing on the HPLC liquid permeate that 40 microlitres (40 μ l) obtain (below, self-contained component A in the future, B component, the liquid permeate of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 63 (2) are called " (XII) metabolism solution (A23) "; In addition, will not comprise B component from not comprising component A, the liquid permeate that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 63 (2) is called " (XII) contrast solution (A23) ").Compare with the concentration of the compound (XII) that detects from (XII) contrast solution (A23), the concentration of the compound (XII) that detects from (XII) metabolism solution (A23) is lower.Detect the peak from (XII) metabolism solution (A23) in addition, it does not detect from (XII) contrast solution (A23).The peak elution time of the compound of the described compound (XII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (2) (XII) metabolism solution (A1) matches.

(9) metabolism of the compound (XII) by protein of the present invention (A24)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 49 (2) in embodiment 65 (2), according to embodiment 68 (1) described methods preparations with keep reaction solns.Be similar to embodiment 68 (1), every kind of reaction soln after preparation keeps.Above-mentioned analysis condition 1 time analyzing on the HPLC liquid permeate that 40 microlitres (40 μ l) obtain (below, self-contained component A in the future, B component, the liquid permeate of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 65 (2) are called " (XII) metabolism solution (A24) "; In addition, will not comprise B component from not comprising component A, the liquid permeate that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 65 (2) is called " (XII) contrast solution (A24) ").Compare with the concentration of the compound (XII) that detects from (XII) contrast solution (A24), the concentration of the compound (XII) that detects from (XII) metabolism solution (A24) is lower.Detect the peak from (XII) metabolism solution (A24) in addition, it does not detect from (XII) contrast solution (A24).The peak elution time of the compound of the described compound (XII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (2) (XII) metabolism solution (A1) matches.

(10) metabolism of the compound (XII) by protein of the present invention (A25)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 49 (2) in embodiment 67 (2), according to embodiment 68 (1) described methods preparations with keep reaction solns.Be similar to embodiment 68 (1), every kind of reaction soln after preparation keeps.Above-mentioned analysis condition 1 time analyzing on the HPLC liquid permeate that 40 microlitres (40 μ l) obtain (below, self-contained component A in the future, B component, the liquid permeate of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 67 (2) are called " (XII) metabolism solution (A25) "; In addition, will not comprise B component from not comprising component A, the liquid permeate that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 67 (2) is called " (XII) contrast solution (A25) ").Compare with the concentration of the compound (XII) that detects from (XII) contrast solution (A25), the concentration of the compound (XII) that detects from (XII) metabolism solution (A25) is lower.Detect the peak from (XII) metabolism solution (A25) in addition, it does not detect from (XII) contrast solution (A25).The peak elution time of the compound of the described compound (XII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (2) (XII) metabolism solution (A1) matches.

(11) metabolism of the compound (XIII) by protein of the present invention (A17)

Except using 12.5ppm compound (XIII) rather than 12.5ppm compound (XII), according to embodiment 68 (2) described method preparation and maintenance reaction solns.Be similar to embodiment 68 (1), every kind of reaction soln after preparation keeps.Above-mentioned analysis condition 1 time analyzing on the HPLC liquid permeate that 40 microlitres (40 μ l) obtain (below, self-contained component A in the future, B component, the liquid permeate of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 51 (2) are called " (XIII) metabolism solution (A17) "; In addition, will not comprise B component from not comprising component A, the liquid permeate that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 51 (2) is called " (XIII) contrast solution (A17) ").Compare with the concentration of the compound (XIII) that detects from (XIII) contrast solution (A17), the concentration of the compound (XIII) that detects from (XIII) metabolism solution (A17) is lower.Detect the peak from (XIII) metabolism solution (A17) in addition, it does not detect from (XIII) contrast solution (A17).The peak elution time of the compound of the described compound (XIII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (3) (XIII) metabolism solution (A1) matches.

(12) metabolism of the compound (XIII) by protein of the present invention (A18)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 51 (2) in embodiment 53 (2), according to embodiment 68 (11) described methods preparations with keep reaction solns.Be similar to embodiment 68 (1), every kind of reaction soln after preparation keeps.Analysis condition 1 time analyzing on the HPLC liquid permeate that 40 microlitres (40 μ l) obtain (below, self-contained component A in the future, B component, the liquid permeate of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 53 (2) are called " (XIII) metabolism solution (A18) "; In addition, will not comprise B component from not comprising component A, the liquid permeate that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 53 (2) is called " (XIII) contrast solution (A18) ").Compare with the concentration of the compound (XIII) that detects from (XIII) contrast solution (A18), the concentration of the compound (XIII) that detects from (XIII) metabolism solution (A18) is lower.Detect the peak from (XIII) metabolism solution (A18) in addition, it does not detect from (XIII) contrast solution (A18).The peak elution time of the compound of the described compound (XIII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (3) (XIII) metabolism solution (A1) matches.

(13) metabolism of the compound (XIII) by protein of the present invention (A19)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 51 (2) in embodiment 55 (2), according to embodiment 68 (11) described methods preparations with keep reaction solns.Be similar to embodiment 68 (1), every kind of reaction soln after preparation keeps.Analysis condition 1 time analyzing on the HPLC liquid permeate that 40 microlitres (40 μ l) obtain (below, self-contained component A in the future, B component, the liquid permeate of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 55 (2) are called " (XIII) metabolism solution (A19) "; In addition, will not comprise B component from not comprising component A, the liquid permeate that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 55 (2) is called " (XIII) contrast solution (A19) ").Compare with the concentration of the compound (XIII) that detects from (XIII) contrast solution (A19), the concentration of the compound (XIII) that detects from (XIII) metabolism solution (A19) is lower.Detect the peak from (XIII) metabolism solution (A19) in addition, it does not detect from (XIII) contrast solution (A19).The peak elution time of the compound of the described compound (XIII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (3) (XIII) metabolism solution (A1) matches.

(14) metabolism of the compound (XIII) by protein of the present invention (A20)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 51 (2) in embodiment 57 (2), according to embodiment 68 (11) described methods preparations with keep reaction solns.Be similar to embodiment 68 (1), every kind of reaction soln after preparation keeps.Analysis condition 1 time analyzing on the HPLC liquid permeate that 40 microlitres (40 μ l) obtain (below, self-contained component A in the future, B component, the liquid permeate of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 57 (2) are called " (XIII) metabolism solution (A20) "; In addition, will not comprise B component from not comprising component A, the liquid permeate that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 57 (2) is called " (XIII) contrast solution (A20) ").Compare with the concentration of the compound (XIII) that detects from (XIII) contrast solution (A20), the concentration of the compound (XIII) that detects from (XIII) metabolism solution (A20) is lower.Detect the peak from (XIII) metabolism solution (A20) in addition, it does not detect from (XIII) contrast solution (A20).The peak elution time of the compound of the described compound (XIII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (3) (XIII) metabolism solution (A1) matches.

(15) metabolism of the compound (XIII) by protein of the present invention (A21)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 51 (2) in embodiment 59 (2), according to embodiment 68 (11) described methods preparations with keep reaction solns.Be similar to embodiment 68 (1), every kind of reaction soln after preparation keeps.Analysis condition 1 time analyzing on the HPLC liquid permeate that 40 microlitres (40 μ l) obtain (below, self-contained component A in the future, B component, the liquid permeate of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 59 (2) are called " (XIII) metabolism solution (A21) "; In addition, will not comprise B component from not comprising component A, the liquid permeate that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 59 (2) is called " (XIII) contrast solution (A21) ").Compare with the concentration of the compound (XIII) that detects from (XIII) contrast solution (A21), the concentration of the compound (XIII) that detects from (XIII) metabolism solution (A21) is lower.Detect the peak from (XIII) metabolism solution (A21) in addition, it does not detect from (XIII) contrast solution (A21).The peak elution time of the compound of the described compound (XIII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (3) (XIII) metabolism solution (A1) matches.

(16) metabolism of the compound (XIII) by protein of the present invention (A23)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 51 (2) in embodiment 63 (2), according to embodiment 68 (11) described methods preparations with keep reaction solns.Be similar to embodiment 68 (1), every kind of reaction soln after preparation keeps.Analysis condition 1 time analyzing on the HPLC liquid permeate that 40 microlitres (40 μ l) obtain (below, self-contained component A in the future, B component, the liquid permeate of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 63 (2) are called " (XIII) metabolism solution (A23) "; In addition, will not comprise B component from not comprising component A, the liquid permeate that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 63 (2) is called " (XIII) contrast solution (A23) ").Compare with the concentration of the compound (XIII) that detects from (XIII) contrast solution (A23), the concentration of the compound (XIII) that detects from (XIII) metabolism solution (A23) is lower.Detect the peak from (XIII) metabolism solution (A23) in addition, it does not detect from (XIII) contrast solution (A23).The peak elution time of the compound of the described compound (XIII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (3) (XIII) metabolism solution (A1) matches.

(17) metabolism of the compound (XIII) by protein of the present invention (A25)

Except use the supernatant liquor fraction that supernatant liquor fraction that 20 μ l reclaim rather than 20 μ l reclaim in embodiment 51 (2) in embodiment 67 (2), according to embodiment 68 (11) described methods preparations with keep reaction solns.Be similar to embodiment 68 (1), every kind of reaction soln after preparation keeps.Analysis condition 1 time analyzing on the HPLC liquid permeate that 40 microlitres (40 μ l) obtain (below, self-contained component A in the future, B component, the liquid permeate of the reaction soln of the supernatant liquor fraction that component C and 20 μ l reclaim in embodiment 67 (2) are called " (XIII) metabolism solution (A25) "; In addition, will not comprise B component from not comprising component A, the liquid permeate that does not comprise component C and be not included in the reaction soln of the supernatant liquor fractions that reclaim among the embodiment 67 (2) is called " (XIII) contrast solution (A25) ").Compare with the concentration of the compound (XIII) that detects from (XIII) contrast solution (A25), the concentration of the compound (XIII) that detects from (XIII) metabolism solution (A25) is lower.Detect the peak from (XIII) metabolism solution (A25) in addition, it does not detect from (XIII) contrast solution (A25).The peak elution time of the compound of the described compound (XIII) little 14 that elution time and the mass ratio at the above peak of HPLC detects in embodiment 41 (3) (XIII) metabolism solution (A1) matches.

Embodiment 69 DNA wherein of the present invention (A1) (A2), is the hybridization of probe (A3) or (A4)

(1) probe preparation

Carry out PCR according to embodiment 30 (1) described methods.Yet, as template, the described 50ng chromosomal DNA of chromosomal DNA that does not produce look streptomycete IFO 12735 that use 10ng prepares in embodiment 26 (1) rather than the abortion within the first month of pregnancy look streptomycete IFO 12898 that in embodiment 3 (1), prepares.As primer, use oligonucleotide with nucleotide sequence shown in the SEQ ID NO:328 and oligonucleotide with nucleotide sequence shown in the SEQ ID NO:329.The DNA of recovery by described pcr amplification is to produce the probe with nucleotide sequence shown in the SEQ ID NO:109 (hereinafter referred to as " probe of DIG mark (A4) ") with the digoxigenin mark.

(2) preparation of plasmid solution

Chromosomal DNA by the black walnut streptomycete IFO 13445 that uses Advantage-GC genome polysaccharase mixture (Clontech Company) and will prepare among embodiment 31 (1) is used as template and carries out PCR.As primer, use oligonucleotide with nucleotide sequence shown in the SEQ ID NO:330 and oligonucleotide with nucleotide sequence shown in the SEQ ID NO:331.By adding 2 kinds every kind primer that amounts to 200nM, 10ng chromosomal DNA, the 4.0 μ l dNTP mixture (mixtures of every kind of 2.5mM of 4 kinds of dNTP; Clontech company), 10.0 μ l 5xGC damping fluids, 2.2 μ l 25mMMg (OAc) ₂, 10.0 μ l 5M GC-Melt and 1.0 μ l Advantage-GC genome polysaccharase mixture (Clontech company) and distilled water, the PCR reaction soln amounts to 50 μ l.The reaction conditions of PCR is to remain on 94 ℃ after 1 minute; Repeat 7 circulations, circulation comprise remain on 94 ℃ 10 seconds, then 72 ℃ 3 minutes; Repeat 36 circulations, circulation comprise 94 ℃ 10 seconds, then 67 ℃ 3 minutes; Remain on then 67 ℃ 7 minutes.By using QIA fast PCR purification kit (Qiagen company) purify DNA from the PCR reaction soln according to the incidental specification sheets of described test kit.According to incidental handbook the DNA that obtains is connected to TA cloning vector pCR2.1 (Invitrogen company) and imports intestinal bacteria TOP10F ' (Invitrogen company).Use QIAprep Spin Miniprep test kit (Qiagen company) preparation plasmid DNA from the intestinal bacteria transformant that obtains to contain the plasmid solution of DNA of the present invention (A11) with acquisition.

Similarly, the chromosomal DNA by Tianjin island chain mould IFO 13782 that will prepare in embodiment 33 (1) carries out PCR as template with by oligonucleotide that will have nucleotide sequence shown in the SEQ ID NO:332 and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:333 as primer.To be connected to carrier by the DNA that PCR obtains above similar.Transformed into escherichia coli then.The preparation plasmid is to obtain to contain the plasmid solution of DNA of the present invention (A12) from the intestinal bacteria transformant that obtains.

Similarly, the chromosomal DNA by the hot lightskyblue streptomycete IFO 14273t that will prepare in embodiment 35 (1) carries out PCR as template with by oligonucleotide that will have nucleotide sequence shown in the SEQ ID NO:331 and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:334 as primer.To be connected to carrier by the DNA that PCR obtains above similar.Transformed into escherichia coli then.The preparation plasmid is to obtain to contain the plasmid solution of DNA of the present invention (A13) from the intestinal bacteria transformant that obtains.

Similarly, the chromosomal DNA that produces look streptomycete IFO 13673t by the pelletizing that will prepare in embodiment 37 (1) carries out PCR as template with by oligonucleotide that will have nucleotide sequence shown in the SEQ ID NO:330 and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:331 as primer.To be connected to carrier by the DNA that PCR obtains above similar.Transformed into escherichia coli then.The preparation plasmid is to obtain to contain the plasmid solution of DNA of the present invention (A14) from the intestinal bacteria transformant that obtains.

Similarly, the chromosomal DNA that produces look streptomycete IFO 12444 by the olive that will prepare in embodiment 39 (1) carries out PCR as template with by oligonucleotide that will have nucleotide sequence shown in the SEQ ID NO:330 and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:331 as primer.To be connected to carrier by the DNA that PCR obtains above similar.Transformed into escherichia coli then.The preparation plasmid is to obtain to contain the plasmid solution of DNA of the present invention (A15) from the intestinal bacteria transformant that obtains.

Similarly, the chromosomal DNA by the decorative chains mould IFO 13069t that will prepare in embodiment 48 (1) carries out PCR as template with by oligonucleotide that will have nucleotide sequence shown in the SEQ ID NO:335 and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:336 as primer.To be connected to carrier by the DNA that PCR obtains above similar.Transformed into escherichia coli then.The preparation plasmid is to obtain to contain the plasmid solution of DNA of the present invention (A16) from the intestinal bacteria transformant that obtains.

Similarly, the chromosomal DNA by the streptomyces griseus ATCC 10137 that will prepare in embodiment 50 (1) carries out PCR as template with by oligonucleotide that will have nucleotide sequence shown in the SEQ ID NO:335 and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:336 as primer.To be connected to carrier by the DNA that PCR obtains above similar.Transformed into escherichia coli then.The preparation plasmid is to obtain to contain the plasmid solution of DNA of the present invention (A17) from the intestinal bacteria transformant that obtains.

Similarly, carry out PCR as template with by oligonucleotide that will have nucleotide sequence shown in the SEQ ID NO:330 and oligonucleotide as primer by the chromosomal DNA that does not produce look streptomycete IFO 12735 that will in embodiment 52 (1), prepare with nucleotide sequence shown in the SEQ ID NO:331.To be connected to carrier by the DNA that PCR obtains above similar.Transformed into escherichia coli then.The preparation plasmid is to obtain to contain the plasmid solution of DNA of the present invention (A18) from the intestinal bacteria transformant that obtains.

Similarly, the chromosomal DNA by the streptomyces griseus IFO 13849T that will prepare in embodiment 54 (1) carries out PCR as template with by oligonucleotide that will have nucleotide sequence shown in the SEQ ID NO:333 and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:335 as primer.To be connected to carrier by the DNA that PCR obtains above similar.Transformed into escherichia coli then.The preparation plasmid is to obtain to contain the plasmid solution of DNA of the present invention (A19) from the intestinal bacteria transformant that obtains.

Similarly, the chromosomal DNA by the wool streptomycete IFO 12787T that will prepare in embodiment 56 (1) carries out PCR as template with by oligonucleotide that will have nucleotide sequence shown in the SEQ ID NO:331 and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:337 as primer.To be connected to carrier by the DNA that PCR obtains above similar.Transformed into escherichia coli then.The preparation plasmid is to obtain to contain the plasmid solution of DNA of the present invention (A20) from the intestinal bacteria transformant that obtains.

Similarly, the chromosomal DNA by the three damp streptomycete IFO 13855T that will prepare in embodiment 58 (1) carries out PCR as template with by oligonucleotide that will have nucleotide sequence shown in the SEQ ID NO:331 and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:338 as primer.To be connected to carrier by the DNA that PCR obtains above similar.Transformed into escherichia coli then.The preparation plasmid is to obtain to contain the plasmid solution of DNA of the present invention (A21) from the intestinal bacteria transformant that obtains.

Similarly, carry out PCR as template and the oligonucleotide by will having nucleotide sequence shown in the SEQ ID NO:331 and oligonucleotide with nucleotide sequence shown in the SEQ ID NO:339 as primer by the redden chromosomal DNA of streptomycete IFO 13682T of the rose that will in embodiment 62 (1), prepare.To be connected to carrier by the DNA that PCR obtains above similar.Transformed into escherichia coli then.The preparation plasmid is to obtain to contain the plasmid solution of DNA of the present invention (A23) from the intestinal bacteria transformant that obtains.

Similarly, the chromosomal DNA by the Regensburg streptomycete IFO 13446T that will prepare in embodiment 66 (1) carries out PCR as template with by oligonucleotide that will have nucleotide sequence shown in the SEQ ID NO:331 and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:339 as primer.To be connected to carrier by the DNA that PCR obtains above similar.Transformed into escherichia coli then.The preparation plasmid is to obtain to contain the plasmid solution of DNA of the present invention (A25) from the intestinal bacteria transformant that obtains.

In addition, similarly, the chromosomal DNA by the pale asphyxia streptomycete IFO13434T that will prepare in embodiment 60 (1) carries out PCR as template with by oligonucleotide that will have nucleotide sequence shown in the SEQ ID NO:340 and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:341 as primer.To be connected to carrier by the DNA that PCR obtains above similar.Transformed into escherichia coli then.The preparation plasmid is to obtain to contain the plasmid solution of DNA of the present invention (A22) from the intestinal bacteria transformant that obtains.

Similarly, the chromosomal DNA of ground, the Shandong by will preparing in embodiment 64 (1) streptomycete IFO 15875T carries out PCR as template with by oligonucleotide that will have nucleotide sequence shown in the SEQ ID NO:342 and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:343 as primer.To be connected to carrier by the DNA that PCR obtains above similar.Transformed into escherichia coli then.The preparation plasmid is to obtain to contain the plasmid solution of DNA of the present invention (A24) from the intestinal bacteria transformant that obtains.

(2) dot hybridization

Will about 100ng and the various plasmid traces that in embodiment 69 (2), prepare of 10ng on HybondN+ nylon membrane (Amersham Biosciences Company).Plasmid is: the plasmid DNA that comprises DNA of the present invention (A11), the plasmid DNA that comprises DNA of the present invention (A12), the plasmid DNA that comprises DNA of the present invention (A13), the plasmid DNA that comprises DNA of the present invention (A14), the plasmid DNA that comprises DNA of the present invention (A15), the plasmid DNA that comprises DNA of the present invention (A16), the plasmid DNA that comprises DNA of the present invention (A17), the plasmid DNA that comprises DNA of the present invention (A18), the plasmid DNA that comprises DNA of the present invention (A19) comprises the plasmid DNA of DNA of the present invention (A20), comprises the plasmid DNA of DNA of the present invention (A21), the plasmid DNA that comprises DNA of the present invention (A23) comprises the plasmid DNA of DNA of the present invention (A25).The film that UV-light point to is obtained with transilluminator 5 minutes.

Hybridize and detect according to embodiment 30 (2) described methods.The probe that will prepare in embodiment 30 (1) is 100 ℃ of maintenances 5 minutes with then fast in cooled on ice.As probe, use the DNA with nucleotide sequence shown in the SEQ ID NO:6 (hereinafter referred to as " probe of DIG mark (A1) ") with the digoxigenin mark, with the DNA with nucleotide sequence shown in the SEQ ID NO:7 (hereinafter referred to as " probe of DIG mark (A2) ") of digoxigenin mark, with the DIG label probe (A4) of the DNA with nucleotide sequence shown in the SEQ ID NO:8 (hereinafter referred to as " probe of DIG mark (A3) ") of digoxigenin mark or generation in embodiment 69 (1).At probe (A1), (A2), (A3) or the situation that any one is used for hybridizing (A4), detect signal for all ingredients of each in 10ng and the above-mentioned plasmid DNA of 100ng with the DIG mark.

In addition, similarly, will about 10ng and the plasmid DNA that contains DNA of the present invention (A22) that in embodiment 69 (2), prepares of 100ng and the plasmid DNA that contains DNA of the present invention (A24) separately trace on Hybond N+ nylon membrane (Amersham Biosciences Company).Hybridize and detect according to embodiment 30 (2).

Embodiment 70 wherein codon uses at the preparation of expressing the DNA of the present invention (A23) (hereinafter referred to as " DNA of the present invention (A23) S ") that adjusts in soybean

(1) preparation of DNA of the present invention (A23) S

Have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:346 and oligonucleotide as primer by use, carry out PCR with Pyrobest archaeal dna polymerase (Takara Shuzo Company) according to incidental handbook with nucleotide sequence shown in the SEQ ID NO:367.The template of the PCR that the aliquot of the PCR product that obtains is carried out as primer as the oligonucleotide that uses the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:345 similarly and have a nucleotide sequence shown in the SEQ ID NO:366.In addition, the template of the PCR that the aliquot of PCR product is carried out as primer as the oligonucleotide that uses the oligonucleotide with nucleotide sequence shown in the SEQID NO:344 similarly and have a nucleotide sequence shown in the SEQ ID NO:365.The reaction soln that obtains is called reaction soln 1.

In addition, according to incidental handbook, have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:349 and oligonucleotide with nucleotide sequence shown in the SEQ ID NO:364 as primer by use, (Takara Shuzo Company) carries out PCR with the Pyrobest archaeal dna polymerase.The template of the PCR that the aliquot of the PCR product that obtains is carried out as primer as the oligonucleotide that uses the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:348 similarly and have a nucleotide sequence shown in the SEQ ID NO:363.In addition, the template of the PCR that the aliquot of PCR product is carried out as primer as the oligonucleotide that uses the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:347 similarly and have a nucleotide sequence shown in the SEQ ID NO:362.The reaction soln that obtains is called reaction soln 2.

In addition, according to incidental handbook, have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:352 and oligonucleotide with nucleotide sequence shown in the SEQ ID NO:361 as primer by use, (Takara Shuzo Company) carries out PCR with the Pyrobest archaeal dna polymerase.The template of the PCR that the aliquot of the PCR product that obtains is carried out as primer as the oligonucleotide that uses the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:351 similarly and have a nucleotide sequence shown in the SEQ ID NO:360.In addition, the template of the PCR that the aliquot of PCR product is carried out as primer as the oligonucleotide that uses the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:350 similarly and have a nucleotide sequence shown in the SEQ ID NO:359.The reaction soln that obtains is called reaction soln 3.

In addition, according to incidental handbook, have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:355 and oligonucleotide with nucleotide sequence shown in the SEQ ID NO:358 as primer by use, (Takara Shuzo Company) carries out PCR with the Pyrobest archaeal dna polymerase.The template of the PCR that the aliquot of the PCR product that obtains is carried out as primer as the oligonucleotide that uses the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:354 similarly and have a nucleotide sequence shown in the SEQ ID NO:357.In addition, the template of the PCR that the aliquot of PCR product is carried out as primer as the oligonucleotide that uses the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:353 similarly and have a nucleotide sequence shown in the SEQ ID NO:356.The reaction soln that obtains is called reaction soln 4.

The reaction soln 1 to 4 that so obtains is mixed.According to incidental handbook, be used as template and use oligonucleotide with nucleotide sequence shown in the SEQ ID NO:344 and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:356 as primer by the aliquot with its mixture, (Takara Shuzo Company) carries out PCR with the Pyrobest archaeal dna polymerase.Confirm the nucleotide sequence of the DNA of amplification.Acquisition has the DNA that nucleotide sequence 5 '-cat-3 ' wherein is connected to the sequence in 5 ' terminal upstream of nucleotide sequence shown in the SEQ ID NO:368 and 3 ' the terminal downstream that nucleotide sequence 5 '-aagctt-3 ' is connected to nucleotide sequence shown in the SEQ IDNO:368.

The codon that shows the DNA of the present invention (A23) with nucleotide sequence shown in the SEQ ID NO:232 in table 28 and table 29 uses (GC content 73.10%).The codon that shows soybean in table 24 and table 25 uses (GC content 46.12%).The codon that shows DNA of the present invention (A23) S with nucleotide sequence shown in the SEQ IDNO:368 in table 30 and table 31 uses (52.38% GC content).

Table 28

Codon	％	Codon	％
Codon	％	Codon	％	TTT	0.00	TCT	0.00
TTC	4.01	TCC	1.50	TTT	0.00	TCT	0.00
TTC	4.01	TCC	1.50	TTA	0.00	TCA	0.00
TTG	0.00	TCG	0.50	TTA	0.00	TCA	0.00
TTG	0.00	TCG	0.50	CTT	0.00	CCT	0.00
CTC	4.26	CCC	5.76	CTT	0.00	CCT	0.00
CTC	4.26	CCC	5.76	CTA	0.00	CCA	0.00
CTG	7.77	CCG	2.26	CTA	0.00	CCA	0.00
CTG	7.77	CCG	2.26	ATT	0.00	ACT	0.00
ATC	4.51	ACC	3.76	ATT	0.00	ACT	0.00
ATC	4.51	ACC	3.76	ATA	0.00	ACA	0.00
ATG	2.26	ACG	2.76	ATA	0.00	ACA	0.00
ATG	2.26	ACG	2.76	GTT	0.00	GCT	0.25
GTC	3.51	GCC	9.27	GTT	0.00	GCT	0.25
GTC	3.51	GCC	9.27	GTA	0.00	GCA	0.75
GTG	2.51	GCG	1.75	GTA	0.00	GCA	0.75

Table 29

Codon	％	Codon	％
Codon	％	Codon	％	TAT	0.00	TGT	0.00
TAC	1.00	TGC	0.75	TAT	0.00	TGT	0.00
TAC	1.00	TGC	0.75	TAA	0.25	TGA	0.00
TAG	0.00	TGG	0.75	TAA	0.25	TGA	0.00
TAG	0.00	TGG	0.75	CAT	0.00	CGT	0.50
CAC	2.26	CGC	6.02	CAT	0.00	CGT	0.50
CAC	2.26	CGC	6.02	CAA	0.50	CGA	0.25

CAG	2.51	CGG	3.01
CAG	2.51	CGG	3.01	AAT	0.00	AGT	0.00
AAC	1.00	AGC	1.25	AAT	0.00	AGT	0.00
AAC	1.00	AGC	1.25	AAA	0.25	AGA	0.00
AAG	0.50	AGG	0.50	AAA	0.25	AGA	0.00
AAG	0.50	AGG	0.50	GAT	0.00	GGT	0.98
GAC	7.27	GGC	6.27	GAT	0.00	GGT	0.98
GAC	7.27	GGC	6.27	GAA	1.25	GGA	0.25
GAG	5.26	GGG	1.00	GAA	1.25	GGA	0.25

Table 30

Codon	％	Codon	％
Codon	％	Codon	％	TTT	2.01	TCT	0.75
TTC	2.01	TCC	0.50	TTT	2.01	TCT	0.75
TTC	2.01	TCC	0.50	TTA	1.00	TCA	0.75
TTG	3.01	TCG	0.25	TTA	1.00	TCA	0.75
TTG	3.01	TCG	0.25	CTT	3.26	CCT	3.01
CTC	2.26	CCC	1.50	CTT	3.26	CCT	3.01
CTC	2.26	CCC	1.50	CTA	1.00	CCA	3.01
CTG	1.50	CCG	0.50	CTA	1.00	CCA	3.01
CTG	1.50	CCG	0.50	ATT	2.26	ACT	2.26
ATC	1.25	ACC	1.75	ATT	2.26	ACT	2.26
ATC	1.25	ACC	1.75	ATA	1.00	ACA	2.01
ATG	2.26	ACG	0.50	ATA	1.00	ACA	2.01
ATG	2.26	ACG	0.50	GTT	2.26	GCT	4.51
GTC	1.00	GCC	2.76	GTT	2.26	GCT	4.51
GTC	1.00	GCC	2.76	GTA	0.75	GCA	3.76
GTG	2.01	GCG	1.00	GTA	0.75	GCA	3.76

Table 31

Codon	％	Codon	％
Codon	％	Codon	％	TAT	0.50	TGT	0.25
TAC	0.50	TGC	0.50	TAT	0.50	TGT	0.25
TAC	0.50	TGC	0.50	TAA	0.25	TGA	0.00
TAG	0.00	TGG	0.75	TAA	0.25	TGA	0.00
TAG	0.00	TGG	0.75	CAT	1.25	CGT	1.50
CAC	1.00	CGC	1.25	CAT	1.25	CGT	1.50
CAC	1.00	CGC	1.25	CAA	1.75	CGA	0.75
CAG	1.25	CGG	0.50	CAA	1.75	CGA	0.75
CAG	1.25	CGG	0.50	AAT	0.50	AGT	0.50
AAC	0.50	AGC	0.50	AAT	0.50	AGT	0.50
AAC	0.50	AGC	0.50	AAA	0.25	AGA	3.26
AAG	0.50	AGG	3.01	AAA	0.25	AGA	3.26
AAG	0.50	AGG	3.01	GAT	4.51	GGT	2.26
GAC	2.76	GGC	1.50	GAT	4.51	GGT	2.26
GAC	2.76	GGC	1.50	GAA	3.26	GGA	2.26
GAG	3.26	GGG	1.50	GAA	3.26	GGA	2.26

(2) has the production of the transformed into escherichia coli of protein of the present invention (A23) S

The DNA with nucleotide sequence shown in the SEQ IDNO:368 of acquisition in embodiment 70 (1) with restriction enzyme NdeI and HindIII digestion.To connect NdeI site and the plasmid between the HindIII site (hereinafter referred to as " pKSN1584soy ") that inserts pKSN2 with the DNA that obtains wherein to have nucleotide sequence shown in the SEQ ID NO:368 with the DNA of the acquisition of NdeI and HindIII digestion and plasmid pKSN2.Described plasmid is imported e. coli jm109.The intestinal bacteria transformant that obtains is called JM109/pKSN1584soy.

(3) expression and the described recovery of protein of protein of the present invention (A23) S in intestinal bacteria

Be similar to embodiment 4 (2), cultivate in e. coli jm109/pKSN1584soy that in embodiment 70 (2), obtains and the e. coli jm109/pKSN1584F that in embodiment 63 (1), obtains each.Reclaim cell.The preparation cell pyrolysis liquid.Under embodiment 4 (2) described methods, preparation supernatant liquor fraction from cell pyrolysis liquid (below, will be called " intestinal bacteria pKSN1584soy extract " from the supernatant liquor fraction that e. coli jm109/pKSN1584soy obtains and will be called " intestinal bacteria pKSN1584F extract " from the supernatant liquor fraction that e. coli jm109/pKSN1584F obtains).The amount of the P450 of each the proteinic amount that comprises in intestinal bacteria pKSN1584soy extract can be comparable to and be higher than the amount of the P450 of each the proteinic amount that comprises in intestinal bacteria pKSN1584F extract.

Embodiment 71 wherein codon uses at preparation and the expression of expressing the DNA of the present invention (A25) (hereinafter referred to as " DNA of the present invention (A25) S ") that adjusts in soybean

(1) preparation of DNA of the present invention (A25) S

Have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:371 and oligonucleotide as primer by use, carry out PCR with Pyrobest archaeal dna polymerase (Takara Shuzo Company) according to incidental handbook with nucleotide sequence shown in the SEQ ID NO:392.The template of the PCR that the aliquot of the PCR product that obtains is carried out as primer as the oligonucleotide that uses the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:370 similarly and have a nucleotide sequence shown in the SEQ ID NO:391.In addition, the template of the PCR that the aliquot of PCR product is carried out as primer as the oligonucleotide that uses the oligonucleotide with nucleotide sequence shown in the SEQID NO:369 similarly and have a nucleotide sequence shown in the SEQ ID NO:390.The reaction soln that obtains is called reaction soln 1.

In addition, according to incidental handbook, have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:374 and oligonucleotide with nucleotide sequence shown in the SEQ ID NO:389 as primer by use, (Takara Shuzo Company) carries out PCR with the Pyrobest archaeal dna polymerase.The template of the PCR that the aliquot of the PCR product that obtains is carried out as primer as the oligonucleotide that uses the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:373 similarly and have a nucleotide sequence shown in the SEQ ID NO:383.In addition, the template of the PCR that the aliquot of PCR product is carried out as primer as the oligonucleotide that uses the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:372 similarly and have a nucleotide sequence shown in the SEQ ID NO:387.The reaction soln that obtains is called reaction soln 2.

In addition, according to incidental handbook, have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:377 and oligonucleotide with nucleotide sequence shown in the SEQ ID NO:386 as primer by use, (Takara Shuzo Company) carries out PCR with the Pyrobest archaeal dna polymerase.The template of the PCR that the aliquot of the PCR product that obtains is carried out as primer as the oligonucleotide that uses the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:376 similarly and have a nucleotide sequence shown in the SEQ ID NO:385.In addition, the template of the PCR that the aliquot of PCR product is carried out as primer as the oligonucleotide that uses the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:375 similarly and have a nucleotide sequence shown in the SEQ ID NO:384.The reaction soln that obtains is called reaction soln 3.

In addition, according to incidental handbook, have the oligonucleotide of nucleotide sequence shown in the SEQ ID NO:380 and oligonucleotide with nucleotide sequence shown in the SEQ ID NO:383 as primer by use, (Takara Shuzo Company) carries out PCR with the Pyrobest archaeal dna polymerase.The template of the PCR that the aliquot of the PCR product that obtains is carried out as primer as the oligonucleotide that uses the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:379 similarly and have a nucleotide sequence shown in the SEQ ID NO:382.In addition, the template of the PCR that the aliquot of PCR product is carried out as primer as the oligonucleotide that uses the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:378 similarly and have a nucleotide sequence shown in the SEQ ID NO:381.The reaction soln that obtains is called reaction soln 4.

The reaction soln 1 to 4 that so obtains is mixed.According to incidental handbook, be used as template and use oligonucleotide with nucleotide sequence shown in the SEQ ID NO:369 and the oligonucleotide with nucleotide sequence shown in the SEQ ID NO:381 as primer by the aliquot with its mixture, (Takara Shuzo Company) carries out PCR with the Pyrobest archaeal dna polymerase.Confirm the nucleotide sequence of the DNA of amplification.Acquisition has the DNA that nucleotide sequence 5 '-cat-3 ' wherein is connected to the sequence in 5 ' terminal upstream of nucleotide sequence shown in the SEQ ID NO:393 and 3 ' the terminal downstream that nucleotide sequence 5 '-aagctt-3 ' is connected to nucleotide sequence shown in the SEQ IDNO:393.

The codon that shows the DNA of the present invention (A25) with nucleotide sequence shown in the SEQ ID NO:234 in table 32 and table 33 uses (GC content 71.93%).The codon that shows soybean in table 24 and table 25 uses (GC content 46.12%).The codon that shows DNA of the present invention (A25) S with nucleotide sequence shown in the SEQ IDNO:393 in table 34 and table 35 uses (52.05% GC content).

Table 32

Codon	％	Codon	％
Codon	％	Codon	％	TTT	0.00	TCT	0.00
TTC	3.76	TCC	1.25	TTT	0.00	TCT	0.00
TTC	3.76	TCC	1.25	TTA	0.00	TCA	0.25
TTG	0.00	TCG	0.75	TTA	0.00	TCA	0.25
TTG	0.00	TCG	0.75	CTT	0.00	CCT	0.25
CTC	4.01	CCC	4.01	CTT	0.00	CCT	0.25
CTC	4.01	CCC	4.01	CTA	0.00	CCA	0.25
CTG	9.52	CCG	2.76	CTA	0.00	CCA	0.25
CTG	9.52	CCG	2.76	ATT	0.00	ACT	0.25
ATC	4.26	ACC	4.01	ATT	0.00	ACT	0.25
ATC	4.26	ACC	4.01	ATA	0.25	ACA	0.00
ATG	2.26	ACG	1.75	ATA	0.25	ACA	0.00
ATG	2.26	ACG	1.75	GTT	0.00	GCT	0.00
GTC	3.01	GCC	8.52	GTT	0.00	GCT	0.00
GTC	3.01	GCC	8.52	GTA	0.00	GCA	0.50
GTG	2.51	GCG	3.01	GTA	0.00	GCA	0.50

Table 33

Codon	％	Codon	％
Codon	％	Codon	％	TAT	0.00	TGT	0.25
TAC	1.25	TGC	0.50	TAT	0.00	TGT	0.25
TAC	1.25	TGC	0.50	TAA	0.25	TGA	0.00
TAG	0.00	TGG	1.00	TAA	0.25	TGA	0.00
TAG	0.00	TGG	1.00	CAT	0.25	CGT	0.75
CAC	2.26	CGC	5.51	CAT	0.25	CGT	0.75
CAC	2.26	CGC	5.51	CAA	0.00	CGA	1.25

CAG	3.01	CGG	3.26
CAG	3.01	CGG	3.26	AAT	0.00	AGT	0.00
AAC	1.00	AGC	1.00	AAT	0.00	AGT	0.00
AAC	1.00	AGC	1.00	AAA	0.25	AGA	0.25
AAG	1.00	AGG	0.00	AAA	0.25	AGA	0.25
AAG	1.00	AGG	0.00	GAT	0.00	GGT	0.25
GAC	7.52	GGC	4.76	GAT	0.00	GGT	0.25
GAC	7.52	GGC	4.76	GAA	1.00	GGA	0.25
GAG	4.76	GGG	1.25	GAA	1.00	GGA	0.25

Table 34

Codon	％	Codon	％
Codon	％	Codon	％	TTT	1.75	TCT	1.25
TTC	2.01	TCC	0.50	TTT	1.75	TCT	1.25
TTC	2.01	TCC	0.50	TTA	1.25	TCA	0.50
TTG	3.26	TCG	0.00	TTA	1.25	TCA	0.50
TTG	3.26	TCG	0.00	CTT	3.51	CCT	2.76
CTC	2.51	CCC	1.25	CTT	3.51	CCT	2.76
CTC	2.51	CCC	1.25	CTA	1.25	CCA	2.76
CTG	1.75	CCG	0.50	CTA	1.25	CCA	2.76
CTG	1.75	CCG	0.50	ATT	2.26	ACT	2.01
ATC	1.25	ACC	1.75	ATT	2.26	ACT	2.01
ATC	1.25	ACC	1.75	ATA	1.00	ACA	1.75
ATG	2.26	ACG	0.50	ATA	1.00	ACA	1.75
ATG	2.26	ACG	0.50	GTT	2.26	GCT	4.51
GTC	1.00	GCC	2.76	GTT	2.26	GCT	4.51
GTC	1.00	GCC	2.76	GTA	0.50	GCA	3.76
GTG	1.75	GCG	1.00	GTA	0.50	GCA	3.76

Table 35

Codon	％	Codon	％
Codon	％	Codon	％	TAT	0.50	TGT	0.25
TAC	0.75	TGC	0.50	TAT	0.50	TGT	0.25
TAC	0.75	TGC	0.50	TAA	0.25	TGA	0.00
TAG	0.00	TGG	1.00	TAA	0.25	TGA	0.00
TAG	0.00	TGG	1.00	CAT	1.25	CGT	1.75
CAC	1.25	CGC	1.50	CAT	1.25	CGT	1.75
CAC	1.25	CGC	1.50	CAA	1.50	CGA	0.75
CAG	1.50	CGG	0.75	CAA	1.50	CGA	0.75
CAG	1.50	CGG	0.75	AAT	0.50	AGT	0.50
AAC	0.50	AGC	0.50	AAT	0.50	AGT	0.50
AAC	0.50	AGC	0.50	AAA	0.50	AGA	3.26
AAG	0.75	AGG	3.01	AAA	0.50	AGA	3.26
AAG	0.75	AGG	3.01	GAT	4.76	GGT	2.01
GAC	2.76	GGC	1.25	GAT	4.76	GGT	2.01
GAC	2.76	GGC	1.25	GAA	2.76	GGA	2.01
GAG	3.01	GGG	1.25	GAA	2.76	GGA	2.01

(2) has the production of the transformed into escherichia coli of protein of the present invention (A25) S

The DNA with nucleotide sequence shown in the SEQ IDNO:393 of acquisition in embodiment 71 (1) with restriction enzyme NdeI and HindIII digestion.To connect NdeI site and the plasmid between the HindIII site (hereinafter referred to as " pKSN1609soy ") that inserts pKSN2 with the DNA that obtains wherein to have nucleotide sequence shown in the SEQ ID NO:393 with the DNA of the acquisition of NdeI and HindIII digestion and plasmid pKSN2.Described plasmid is imported e. coli jm109.The intestinal bacteria transformant that obtains is called JM109/pKSN1609soy.

(3) expression and the described recovery of protein of protein of the present invention (A25) S in intestinal bacteria

Be similar to embodiment 4 (2), cultivate in e. coli jm109/pKSN1609soy that in embodiment 71 (2), obtains and the e. coli jm109/pKSN1609F that in embodiment 67 (1), obtains each.Reclaim cell.The preparation cell pyrolysis liquid.Under embodiment 4 (2) described methods, preparation supernatant liquor fraction from cell pyrolysis liquid (below, will be called " intestinal bacteria pKSN1609soy extract " from the supernatant liquor fraction that e. coli jm109/pKSN1609soy obtains and will be called " intestinal bacteria pKSN1609F extract " from the supernatant liquor fraction that e. coli jm109/pKSN1609F obtains).The amount of the P450 of each the proteinic amount that comprises in intestinal bacteria pKSN1609soy extract can be comparable to and be higher than the amount of the P450 of each the proteinic amount that comprises in intestinal bacteria pKSN1609F extract.

The preparation of the antibody of the present invention (A) (hereinafter referred to as " antibody of the present invention (A25) ") of embodiment 72 identification protein of the present invention (A25)

(1) preparation of the colibacillary extract of expression protein of the present invention (A25)

According to embodiment 4 (2) described methods, the pre-overnight incubation of e. coli jm109/pKSN1609soy that will in embodiment 71 (2), produce.The culture medium inoculated that obtains is cultivated down to the 1L TB substratum that comprises 50 μ g/ml penbritins and at 26 ℃.Then the 5-amino-laevulic acid is added to the final concentration of 500 μ M,, further cultivate the final concentration that IPTG adds to 1mM.From substratum, reclaim cell, with 0.05MTris-HCl damping fluid (pH7.5) washing be suspended in then in the described damping fluid that 100ml comprises 1mMPMSF.The cell culture medium that obtains in work output 5, is carried out ultrasonoscope (Sonifer (Branson Sonic PowerCompany)) 3 times under the condition of space factor 30%, each 10 minutes, so that obtain cell pyrolysis liquid.With cell pyrolysis liquid centrifugal (9,000xg, 10 minutes) back reclaim supernatant liquor and centrifugal (200,000xg, 70 minutes) with reclaim the supernatant liquor fraction (below, the supernatant liquor fraction that will obtain from e. coli jm109/pKSN1609soy is called " intestinal bacteria pKSN11609soy extract ".

(2) purifying of protein of the present invention (A25)

The supernatant liquor fraction (intestinal bacteria pKSN11609soy extract) that will obtain in embodiment 72 (1) is expelled in the Hiload HiLoad 16/10Q Sepharose HP post (AmershamBioscience Company).Next, after the two three propane damping fluids (pH7.0) of the 20mM of 40ml flow to pillar, (gradient of NaCl is 0.00125M/ minute with the NaCl of linear gradient, the NaCl range of concentrations is 0M-0.375M, and flow velocity is 3ml/ minute) the two three propane damping fluids of the 20mM that flows are recovered in the 10ml fraction of wash-out under the NaCl concentration of 0.088M-0.100M with classification.

The fraction that reclaims is carried out PD10 post (Amersham Biosciences Company), comprise proteinic fraction with recovery with two three propane damping fluid (pH7.0) wash-outs of 20mM.Next, described fraction is injected among the MonoQ HR 10/10 (Amersham Biosciences Company).The two three propane damping fluids of 16 milliliters of (16ml) 20mM are flow to pillar.Next, (gradient of NaCl is 0.001042M/ minute with the NaCl of linear gradient, the NaCl range of concentrations is 0M-0.25M, and flow velocity is 4ml/ minute) the two three propane damping fluids of the 20mM that flows are recovered in the 8ml fraction of wash-out under the NaCl concentration of 0.060M-0.069M with classification.

With the two three propane damping fluids (pH7.0) of 20mM with 2.5 times of the fraction dilutions of reclaiming and be injected in the MonoQHR5/5 post (Amersham Biosciences Company).Next, after two three propane damping fluids (pH 7.0) flow to pillar with 2ml 20mM, (gradient of NaCl is 0.008333M/ minute with the NaCl of linear gradient, the NaCl range of concentrations is 0M-0.25M, and flow velocity is 1ml/ minute) the two three propane damping fluids of the 20mM that flows are recovered in the 0.5ml fraction of wash-out under the NaCl concentration of 0.073M-0.077M with classification.

By using " PAG mini Daiichi 10/20 " (DaiichiPure Chemicals company limited) to analyze the fraction of purifying like this, are the fractions that mainly comprise protein of the present invention (A25) to confirm those fractions with SDS-PAGE.

(3) preparation of antibody of the present invention (A25)

Carry out the preparation of antibody of the present invention according to embodiment 44 (3) described methods.Yet, not being to use protein of the present invention (A1), the protein of the present invention (A25) that will obtain in embodiment 72 (2) is used for obtaining to contain the antiserum(antisera) of antibody of the present invention (A25).

Embodiment 73 detects protein of the present invention by antibody of the present invention (A25)

By using the antibody of the present invention (A25) that in embodiment 72 (3), obtains to carry out immunoblotting with every kind of intestinal bacteria extract.Carry out the sds polyacrylamide electrophoresis (40mA, 1 hour) of and the following: the intestinal bacteria pKSN452F extract (containing the 2pmol that has an appointment protein of the present invention (A16)) that in embodiment 49 (2), obtains; The intestinal bacteria pKSN608F extract (containing the 2pmol that has an appointment protein of the present invention (A17)) that in embodiment 51 (2), obtains; The intestinal bacteria pKSN646BF extract (containing the 2pmol that has an appointment protein of the present invention (A18)) that in embodiment 53 (2), obtains; The intestinal bacteria pKSN1502F extract (containing the 2pmol that has an appointment protein of the present invention (A19)) that in embodiment 55 (2), obtains; The intestinal bacteria pKSN1525F extract (containing the 2pmol that has an appointment protein of the present invention (A20)) that in embodiment 57 (2), obtains; The intestinal bacteria pKSN1543BF extract (containing the 2pmol that has an appointment protein of the present invention (A21)) that in embodiment 59 (2), obtains; The intestinal bacteria pKSN1558BF extract (containing the 2pmol that has an appointment protein of the present invention (A22)) that in embodiment 61 (2), obtains; The intestinal bacteria pKSN1584F extract (containing the 2pmol that has an appointment protein of the present invention (A23)) that in embodiment 63 (2), obtains; The intestinal bacteria pKSN1589BF extract (containing the 2pmol that has an appointment protein of the present invention (A24)) that in embodiment 65 (2), obtains; The intestinal bacteria pKSN1609F extract (containing the 0.5pmol that has an appointment protein of the present invention (A25)) that in embodiment 67 (2), obtains; The intestinal bacteria pKSN1584soy extract (containing the 2pmol that has an appointment protein of the present invention (A23)) that in embodiment 70 (3), obtains; The intestinal bacteria pKSN1609soy extract (containing the 0.5pmol that has an appointment protein of the present invention (A25)) that in embodiment 71 (3), obtains; With the intestinal bacteria pKSN2 extract (containing the 0.8mg protein of having an appointment) that in embodiment 67 (2), obtains.According to embodiment 45 described methods the protein transduction in the described gel is moved on on the PDVF film.According to embodiment 45 described methods, the PDVF film that will in embodiment 45, obtain (hereinafter referred to as " PDVF film (A) ") and PDVF film (hereinafter referred to as " PDVF film (B) ") that from aforesaid method, obtains and the antiserum(antisera) reaction that in embodiment 72 (3), obtains.Subsequently, carry out reaction, washing and dyeing with second antibody according to embodiment 45 described methods.On PDVF film (A), detect corresponding to protein of the present invention (A1), (A2), (A3), (A4), (A11), (A12), (A13), (A14) and (A15) and the dyeing of protein of the present invention (A9) and band (A10).On PDVF film (B), detect corresponding to protein of the present invention (A16), (A17), (A18), (A19), (A20), (A21), (A22), (A23), the dyeing of band (A24) and (A25).Do not detect painted band for intestinal bacteria pKSN2 extract (the containing 0.78mg protein) reagent that in embodiment 4 (2), obtains of pvdf membrane (A) with for intestinal bacteria pKSN2 extract (the containing 0.8mg protein) reagent of the acquisition among embodiment 67 (2) of pvdf membrane (B).

Embodiment 74 imports plant with DNA of the present invention (A23) S

(1) is used for the directly structure-part 1 of the chloroplast expression plasmid that comprises DNA of the present invention (A23) S of importing

The plasmid that structure comprises chimeric DNA is as importing the plasmid of plant with particle bombardment with DNA of the present invention (A23) S, and DNA of the present invention (A23) S is right after after the nucleotide sequence of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and does not have a variation of codon frame in the described chimeric DNA.

At first, the DNA that comprises nucleotide sequence shown in the SEQ ID NO:398 by pcr amplification.By the pKSN1584soy that will in embodiment 70 (2), obtain as template with by the oligonucleotide that will form by nucleotide sequence shown in the SEQIDNO:397 be used as primer by the oligonucleotide that nucleotide sequence shown in the SEQ ID NO:398 is formed and carry out PCR.PCR uses KOD-plus (Toyobo Company).According to the following PCR that carries out: under 94 ℃, keep 2 minutes once; 20 circulations, circulation comprise keep 94 ℃ 30 seconds, then 53 ℃ 30 seconds, then 68 ℃ 90 seconds; Remain at last 68 ℃ 3 minutes.By carrying out program MagExtractor-PCR﹠amp according to subsidiary handbook; The DNA that Gel-Clean up (Toyobo Company) reclaims and purifying increases.By handling the DNA that obtains with TaKaRa BKL test kit (Takara ShuzoCompany), with flat endization of DNA and 5 ' terminal phosphateization according to incidental handbook.Recovery comprises the DNA of nucleotide sequence shown in the SEQ ID NO:368.After with SmaI digested plasmid pUC19 (Takara Shuzo Company), use calf intestinal alkaline phosphatase (Takara ShuzoCompany) with 5 ' terminal dephosphorylation.Produce plasmid by connecting dephosphorylized DNA that produces and the DNA that contains nucleotide sequence shown in the SEQ ID NO:368.After the plasmid that obtains with restriction enzyme EcoT22I and SacI digestion, reclaim the DNA that comprises nucleotide sequence shown in the SEQ ID NO:368.After the plasmid pUCrSt657 that in embodiment 16 (2), obtains with restriction enzyme EcoT22I and SacI digestion, the DNA that separates about 2.9kbp, it has the nucleotide sequence that derives from pUC19 and the sequence of soybean (cv.Jack) the RuBPC small subunit chloroplast transit peptides of encoding.The DNA that obtains is connected the pUCrSt1584soy (Figure 54) that comprises chimeric DNA with acquisition with the above-mentioned DNA that comprises nucleotide sequence shown in the SEQ ID NO:368, DNA wherein of the present invention (A23) S is right after after the nucleotide sequence of soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides of encoding and does not have a variation of codon frame.

Digest the plasmid pUCrSt1584soy of acquisition to separate the DNA that comprises nucleotide sequence shown in the SEQ ID NO:368 with restriction enzyme BamHI and SacI.Described DNA is inserted between the BglII restriction enzyme sites of the plasmid pNdG6-Δ T that obtain among the embodiment 16 (2) and the SacI restriction enzyme sites to obtain plasmid pSUM-NdG6-rSt-1584soy (Figure 55), wherein the CR16G6 promotor has been connected to the downstream of chimeric DNA, is right after after the nucleotide sequence of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and does not have a variation of codon frame at DNA described in the chimeric DNA.

Next, plasmid is imported bacillus coli DH 5 alpha competent cell (Takara ShuzoCompany) and selection amicillin resistance cell.In addition, by using BigDye to stop the nucleotide sequence that cycle sequencing easy reaction test kit 3.0 editions (PE Applied Biosystems Company) and dna sequencing instrument 3100 (PE Applied Biosystems Company) mensuration are included in the plasmid in the amicillin resistance bacterial strain.As a result, confirm that plasmid pSUM-NdG6-rSt-1584soy has nucleotide sequence shown in the SEQ ID NO:368.

(2) be used for directly structure-partly (2) of the chloroplast expression plasmid with DNA of the present invention (A23) S of importing

Structure imports DNA of the present invention (A23) S with particle bombardment the plasmid of plant.This plasmid comprises chimeric DNA, and DNA wherein of the present invention (A23) S is right after after 12 amino acid whose nucleotide sequences of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and the mature protein thereafter of encoding and does not have a variation of codon frame.At first, the DNA that comprises nucleotide sequence shown in the SEQ ID NO:368 by pcr amplification.By the pKSN1584soy that will in embodiment 70, obtain as template with by the oligonucleotide that will form by nucleotide sequence shown in the SEQ ID NO:399 be used as primer by the oligonucleotide that nucleotide sequence shown in the SEQ ID NO:398 is formed and carry out PCR.PCR uses KOD-plus (Toyobo Company).According to the following PCR that carries out: under 94 ℃, keep 2 minutes once; 25 circulations, circulation comprise keep 94 ℃ 30 seconds, then 46 ℃ 30 seconds, then 68 ℃ 90 seconds; Remain at last 68 ℃ 3 minutes.By carrying out program MagExtractor-PCR﹠amp according to subsidiary handbook; The DNA that Gel-Cleanup (Toyobo Company) reclaims and purifying increases.By handling the DNA that obtains with TaKaRa BKL test kit (TaKaRaShuzo Company), with the flat endization of DNA with 5 ' terminal phosphateization according to subsidiary handbook.Recovery contains the DNA of nucleotide sequence shown in the SEQ ID NO:368.After the plasmid pKF19 Δ Bs that in embodiment 15 (3), obtains with SmaI digestion, use calf intestinal alkaline phosphatase (TakaraShuzo Company) with 5 ' terminal dephosphorylation.Produce plasmid by connecting dephosphorylized DNA that produces and the DNA that contains nucleotide sequence shown in the SEQ ID NO:368.After the plasmid that obtains with restriction enzyme BspHI and SacI digestion, reclaim the DNA that contains nucleotide sequence shown in the SEQ ID NO:368.Then digest the plasmid pKFrSt12-657 of acquisition in embodiment 16 (3) to separate DNA from plasmid pKFrSt12 with restriction enzyme BspHI and SacI.With described DNA be connected with the DNA that comprises nucleotide sequence shown in the SEQ ID NO:368 with restriction enzyme SacI and BspHI digestion, so that obtain to comprise the plasmid pKFrSt12-1584soy (Figure 56) of chimeric DNA, DNA of the present invention (A23) S is right after after 12 amino acid whose nucleotide sequences of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and the mature protein thereafter of encoding and does not have a variation of codon frame in described chimeric DNA.

Digest the plasmid pKFrSt12-1584soy of acquisition to separate the DNA that comprises nucleotide sequence shown in the SEQ ID NO:368 with restriction enzyme BamHI and SacI.Described DNA is inserted between the BglII restriction enzyme sites of plasmid pNdG6-Δ T and the SacI restriction enzyme sites to obtain plasmid pSUM-NdG6-rSt12-1584soy (Figure 57), wherein the CR16G6 promotor has been connected to the downstream of chimeric DNA, and DNA described in the described chimeric DNA is right after after the nucleotide sequence of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and does not have a variation of codon frame.

Next, plasmid is imported bacillus coli DH 5 alpha competent cell (Takara ShuzoCompany) and selection amicillin resistance cell.In addition, by using BigDye to stop the nucleotide sequence that cycle sequencing easy reaction test kit 3.0 editions (PE Applied Biosystems Company) and dna sequencing instrument 3100 (PE Applied Biosystems Company) mensuration are included in the plasmid in the amicillin resistance bacterial strain.As a result, confirm that plasmid pSUM-NdG6-rSt12-1584soy has nucleotide sequence shown in the SEQ ID NO:368.

(3) DNA of the present invention (A23) S is imported soybean

The globular embryo for preparing soybean (Cultivar: Fayette and Jack) according to embodiment 47 (3) described methods.

Be transplanted to the globular embryo that obtains in the fresh body somatic embryo growth medium and cultivated 2-3 days.According to embodiment 17 (2) described methods, plasmid pSUM-NdG6-rSt-1584soy that will produce in embodiment 74 (1) or the plasmid pSUM-NdG6-rSt12-1584soy that produces in embodiment 74 (2) import those globular embryos.

(4) with Totomycin selective body somatic embryo

Carry out being chosen in the globular embryo after gene imports that obtains among the embodiment 74 (3) according to embodiment 47 (4) described methods with Totomycin.

(5) with compound (II) selective body somatic embryo

Be chosen in the later globular embryo of quiding gene that obtains among the embodiment 74 (3) according to embodiment 47 (5) described method compounds (II).

(6), comply with and cultivate from the plant regeneration of somatic embryo

According to embodiment 47 (6) described methods, carry out the regeneration of plant from the globular embryo of embodiment 74 (4) or 74 (5), selecting.

(7) resistance of herbicidal compound (II) is assessed

Be evaluated at the resistance level of the aftergrowth of acquisition among the embodiment 74 (6) according to embodiment 17 (4) described methods to compound (II).

(8) be used for the structure of the chloroplast expression plasmid that Agrobacterium imports with DNA of the present invention (A23) S

Structure imports DNA of the present invention (A23) S with agrobacterium co-cultivation the plasmid of plant.Separating chimeric DNA, DNA wherein of the present invention (A23) S is right after after the nucleotide sequence of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and does not have a variation of codon frame with restriction enzyme HindIII and EcoRI digested plasmid pSUM-NdG6-rSt-1584soy.Described DNA is inserted among the embodiment 18 between the HindIII restriction site of the above-mentioned binary plasmid carrier pBI121S that obtains and the EcoRI restriction site to obtain plasmid pBI-NdG6-rSt-1584soy (Figure 58).In addition, separating chimeric DNA, DNA wherein of the present invention (A23) S is right after after 12 amino acid whose nucleotide sequences of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and the mature protein thereafter of encoding and does not have a variation of codon frame with restriction enzyme NotI digested plasmid pSUM-NdG6-rSt12-1584soy.This DNA is inserted between the HindIII restriction site of above-mentioned binary plasmid carrier pBI121S and the EcoRI restriction site to obtain plasmid pBI-NdG6-rSt12-1584soy (Figure 59).

(9) DNA of the present invention (A23) S is imported tobacco

Plasmid pBI-NdG6-rSt-1584soy and pBI-NdG6-rSt12-1584soy that use obtains in embodiment 74 (8) import tobacco with agrobacterium co-cultivation with DNA of the present invention (A23) S.

At first, according to embodiment 19 described methods, respectively plasmid pBI-NdG6-rSt-1584soy and pBI-NdG6-rSt12-1584soy are imported agrobacterium tumefaciens lba4404 (ClontechCompany).Separate the transgenosis Agrobacterium that has pBI-NdG6-rSt-1584soy or pBI-NdG6-rSt12-1584soy separately.

Then, the described Agrobacterium that will have plasmid according to embodiment 47 (9) described methods is used for gene is imported tobacco, and the transgene tobacco in the T-DNA zone of pBI-NdG6-rSt-1584soy or pBI-NdG6-rSt12-1584soy has been integrated in acquisition respectively.

(10) use the resistance of the blade of DNA of the present invention (A23) S transgene tobacco to assess

Gather leaf from the transgene tobacco that 35 strains obtain among embodiment 74 (9).Every leaf is divided into several, and wherein every 5-7mm is wide.Blade is planted on the MS nutrient agar of inclusion compound (II) or compound (XII) and in illumination and at room temperature cultivate.After cultivating several days, observe the weeding damage of each blade.In contrast, use the blade of wild-type tobacco.By blade, have the blade of chemical damage and bleach and the keep the score resistance of assessment transgene tobacco of withered blade continuous growth.

Embodiment 75 imports plant with DNA of the present invention (A25) S

(1) is used for the directly structure-part 1 of the chloroplast expression plasmid that comprises DNA of the present invention (A25) S of importing

The plasmid that structure comprises chimeric DNA is as importing the plasmid of plant with particle bombardment with DNA of the present invention (A25) S, and DNA of the present invention (A25) S is right after after the nucleotide sequence of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and does not have a variation of codon frame in the described chimeric DNA.

At first, the DNA that comprises nucleotide sequence shown in the SEQ ID NO:393 by pcr amplification.By the pKSN1609soy that will in embodiment 71 (2), obtain as template with by the oligonucleotide that will form by nucleotide sequence shown in the SEQID NO:400 be used as primer by the oligonucleotide that nucleotide sequence shown in the SEQ ID NO:401 is formed and carry out PCR.PCR uses KOD-plus (Toyobo Company).According to the following PCR that carries out: under 94 ℃, keep 2 minutes once; 20 circulations, circulation comprise keep 94 ℃ 30 seconds, then 53 ℃ 30 seconds, then 68 ℃ 90 seconds; Remain at last 68 ℃ 3 minutes.By carrying out program MagExtractor-PCR﹠amp according to subsidiary handbook; The DNA that Gel-Clean up (Toyobo Company) reclaims and purifying increases.By handling the DNA that obtains with TaKaRa BKL test kit (Takara ShuzoCompany), with flat endization of DNA and 5 ' terminal phosphateization according to incidental handbook.Recovery comprises the DNA of nucleotide sequence shown in the SEQ ID NO:393.After with SmaI digested plasmid pUC19 (Takara Shuzo Company), use calf intestinal alkaline phosphatase (Takara ShuzoCompany) with 5 ' terminal dephosphorylation.Produce plasmid by connecting dephosphorylized DNA that produces and the DNA that contains nucleotide sequence shown in the SEQ ID NO:393.After the plasmid that obtains with restriction enzyme EcoT22I and SacI digestion, reclaim the DNA that comprises nucleotide sequence shown in the SEQ ID NO:393.After the plasmid pUCrSt657 that in embodiment 16 (2), obtains with restriction enzyme EcoT22I and SacI digestion, the DNA that separates about 2.9kbp, it has the nucleotide sequence that derives from pUC19 and the sequence of soybean (cv.Jack) the RuBPC small subunit chloroplast transit peptides of encoding.The DNA that obtains is connected the pUCrSt1609soy (Figure 60) that comprises chimeric DNA with acquisition with the above-mentioned DNA that comprises nucleotide sequence shown in the SEQ ID NO:393, DNA wherein of the present invention (A25) S is right after after the nucleotide sequence of soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides of encoding and does not have a variation of codon frame.

Digest the plasmid pUCrSt1609soy of acquisition to separate the DNA that comprises nucleotide sequence shown in the SEQ ID NO:393 with restriction enzyme BamHI and SacI.Described DNA is inserted between the BglII restriction enzyme sites of plasmid pNdG6-Δ T and the SacI restriction enzyme sites to obtain plasmid pSUM-NdG6-rSt-1609soy (Figure 61), wherein the CR16G6 promotor has been connected to the downstream of chimeric DNA, is right after after the nucleotide sequence of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and does not have a variation of codon frame at DNA described in the chimeric DNA.

Next, plasmid is imported bacillus coli DH 5 alpha competent cell (Takara ShuzoCompany) and selection amicillin resistance cell.In addition, by using BigDye to stop the nucleotide sequence that cycle sequencing easy reaction test kit 3.0 editions (PE Applied Biosystems Company) and dna sequencing instrument 3100 (PE Applied Biosystems Company) mensuration are included in the plasmid in the amicillin resistance bacterial strain.As a result, confirm that plasmid pSUM-NdG6-rSt-1609soy has nucleotide sequence shown in the SEQ ID NO:393.

(2) be used for directly structure-partly (2) of the chloroplast expression plasmid with DNA of the present invention (A25) S of importing

Structure imports DNA of the present invention (A25) S with particle bombardment the plasmid of plant.This plasmid comprises chimeric DNA, and DNA wherein of the present invention (A25) S is right after after 12 amino acid whose nucleotide sequences of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and the mature protein thereafter of encoding and does not have a variation of codon frame.At first, the plasmid pUCrSt1609soy that obtains in embodiment 75 (1) inserts joint EcoT22I-12aa-EcoT22I (Figure 62) at its EcoT22I restriction site, and this joint is by the oligonucleotide that will be made up of nucleotide sequence shown in the SEQ ID NO:402 with by the oligonucleotide that nucleotide sequence shown in the SEQ ID NO:403 the is formed acquisition of annealing.Acquisition comprises the plasmid pUCrSt12-1609soy (Figure 63) of chimeric DNA, and DNA of the present invention (A25) S is right after after 12 amino acid whose nucleotide sequences of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and the mature protein thereafter of encoding and does not have a variation of codon frame in described chimeric DNA.

Digest the plasmid pUCrSt12-1609soy of acquisition to separate the DNA that comprises nucleotide sequence shown in the SEQ ID NO:393 with restriction enzyme BamHI and SacI.Described DNA is inserted between the BglII restriction enzyme sites of the plasmid pNdG6-Δ T that obtain among the embodiment 16 (2) and the SacI restriction enzyme sites to obtain plasmid pSUM-NdG6-rSt12-1609soy (Figure 64), wherein the CR16G6 promotor has been connected to the downstream of chimeric DNA, and DNA described in the described chimeric DNA is right after after the nucleotide sequence of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and does not have a variation of codon frame.

Next, plasmid is imported bacillus coli DH 5 alpha competent cell (Takara ShuzoCompany) and selection amicillin resistance cell.In addition, by using BigDye to stop the nucleotide sequence that cycle sequencing easy reaction test kit 3.0 editions (PE Applied Biosystems Company) and dna sequencing instrument 3100 (PE Applied Biosystems Company) mensuration are included in the plasmid in the amicillin resistance bacterial strain.As a result, confirm that plasmid pSUM-NdG6-rSt12-1609soy has nucleotide sequence shown in the SEQ ID NO:393.

(3) DNA of the present invention (A23) S is imported soybean

Be transplanted to the globular embryo that obtains in the fresh body somatic embryo growth medium and cultivated 2-3 days.According to embodiment 17 (2) described methods, plasmid pSUM-NdG6-rSt-1609soy that will produce in embodiment 75 (1) or the plasmid pSUM-NdG6-rSt12-1609soy that produces in embodiment 75 (2) import those globular embryos.

(4) with Totomycin selective body somatic embryo

Carry out being chosen in the globular embryo after gene imports that obtains among the embodiment 75 (3) according to embodiment 47 (4) described methods with Totomycin.

(5) with compound (II) selective body somatic embryo

Be chosen in the later globular embryo of quiding gene that obtains among the embodiment 75 (3) according to embodiment 47 (5) described method compounds (II).

(6), comply with and cultivate from the plant regeneration of somatic embryo

(7) resistance of herbicidal compound (II) is assessed

Be evaluated at the resistance level of the aftergrowth of acquisition among the embodiment 75 (6) according to embodiment 17 (4) described methods to compound (II).

(8) be used for the structure of the chloroplast expression plasmid that Agrobacterium imports with DNA of the present invention (A25) S

Structure imports DNA of the present invention (A25) S with agrobacterium co-cultivation the plasmid of plant.Obtaining chimeric DNA, DNA wherein of the present invention (A25) S is right after after the nucleotide sequence of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and does not have a variation of codon frame with restriction enzyme HindIII and EcoRI digested plasmid pSUM-NdG6-rSt-1609soy.Described DNA is inserted among the embodiment 18 between the HindIII restriction site of the binary plasmid carrier pBI121S that obtains and the EcoRI restriction site to obtain plasmid pBI-NdG6-rSt-1609soy (Figure 65).In addition, separating chimeric DNA, DNA wherein of the present invention (A25) S is right after after 12 amino acid whose nucleotide sequences of coding soybean (cv.Jack) RuBPC small subunit chloroplast transit peptides and the mature protein thereafter of encoding and does not have a variation of codon frame with restriction enzyme NotI digested plasmid pSUM-NdG6-rSt12-1609soy.This DNA is inserted between the HindIII restriction site of above-mentioned binary plasmid carrier pBI121S and the EcoRI restriction site to obtain plasmid pBI-NdG6-rSt12-1609soy (Figure 66).

(9) DNA of the present invention (A23) S is imported tobacco

Plasmid pBI-NdG6-rSt-1609soy and pBI-NdG6-rSt12-1609soy that use obtains in embodiment 75 (8) import tobacco with agrobacterium co-cultivation with DNA of the present invention (A25) S.

At first, according to embodiment 19 described methods, respectively plasmid pBI-NdG6-rSt-1609soy and pBI-NdG6-rSt12-1609soy are imported agrobacterium tumefaciens lba4404 (C1ontechCompany).Separate the transgenosis Agrobacterium that has pBI-NdG6-rSt-1609soy or pBI-NdG6-rSt12-1609soy separately.

Then, the described Agrobacterium that will have plasmid according to embodiment 47 (9) described methods is used for gene is imported tobacco, and the transgene tobacco in the T-DNA zone of pBI-NdG6-rSt-1609soy or pBI-NdG6-rSt12-1609soy has been integrated in acquisition respectively.

(10) use the resistance of the blade of DNA of the present invention (A25) S transgene tobacco to assess

Gather leaf from the transgene tobacco that among embodiment 75 (9), obtains.According to embodiment 74 (10) described methods, this leaf is used to assess the resistance of transgene tobacco to compound (II) or compound (XII).

Industrial usability

According to the present invention, can provide to have the protein that metabolism PPO suppresses herbicides compounds and this compound changed into the ability of the compound that hangs down activity of weeding; This protein DNA of encoding; Plant with the anti-herbicides compounds of expressing this protein.

Sequence independence text

SEQ ID NO：35

The Oligonucleolide primers that designs for PCR

SEQ ID NO：36

The Oligonucleolide primers that designs for PCR

SEQ ID NO：37

The Oligonucleolide primers that designs for PCR

SEQ ID NO：38

The Oligonucleolide primers that designs for PCR

SEQ ID NO：39

The Oligonucleolide primers that designs for PCR

SEQ ID NO：40

The Oligonucleolide primers that designs for PCR

SEQ ID NO：41

The Oligonucleolide primers that designs for PCR

SEQ ID NO：42

The Oligonucleolide primers that designs for PCR

SEQ ID NO：43

The Oligonucleolide primers that designs for PCR

SEQ ID NO：44

The Oligonucleolide primers that designs for PCR

SEQ ID NO：45

The Oligonucleolide primers that designs for PCR

SEQ ID NO：46

The Oligonucleolide primers that designs for PCR

SEQ ID NO：47

The Oligonucleolide primers that designs for PCR

SEQ ID NO：48

The Oligonucleolide primers that designs for PCR

SEQ ID NO：49

The Oligonucleolide primers that designs for PCR

SEQ ID NO：50

The Oligonucleolide primers that designs for PCR

SEQ ID NO：51

The Oligonucleolide primers that designs for PCR

SEQ ID NO：52

The Oligonucleolide primers that designs for PCR

SEQ ID NO：53

The Oligonucleolide primers that designs for PCR

SEQ ID NO：54

The Oligonucleolide primers that designs for PCR

SEQ ID NO：55

The Oligonucleolide primers that designs for PCR

SEQ ID NO：56

The Oligonucleolide primers that designs for PCR

SEQ ID NO：57

The Oligonucleolide primers that designs for PCR

SEQ ID NO：58

The Oligonucleolide primers that designs for PCR

SEQ ID NO：59

The Oligonucleolide primers that designs for PCR

SEQ ID NO：60

The Oligonucleolide primers that designs for PCR

SEQ ID NO：61

The Oligonucleolide primers that designs for PCR

SEQ ID NO：62

The Oligonucleolide primers that designs for PCR

SEQ ID NO：63

The Oligonucleolide primers that designs for PCR

SEQ ID NO：64

The Oligonucleolide primers that designs for PCR

SEQ ID NO：65

The Oligonucleolide primers that designs for PCR

SEQ ID NO：66

The Oligonucleolide primers that designs for PCR

SEQ ID NO：67

The Oligonucleolide primers that designs for PCR

SEQ ID NO：68

The Oligonucleolide primers that designs for PCR

SEQ ID NO：70

The Oligonucleolide primers that designs for PCR

SEQ ID NO：71

The Oligonucleolide primers that designs for PCR

SEQ ID NO：72

The Oligonucleolide primers that designs for PCR

SEQ ID NO：73

The Oligonucleolide primers that designs for PCR

SEQ ID NO：74

The Oligonucleolide primers that designs for PCR

SEQ ID NO：75

The Oligonucleolide primers that designs for PCR

SEQ ID NO：76

The Oligonucleolide primers that designs for PCR

SEQ ID NO：77

The Oligonucleolide primers that designs for PCR

SEQ ID NO：79

The Oligonucleolide primers that designs for PCR

SEQ ID NO：80

The Oligonucleolide primers that designs for PCR

SEQ ID NO：81

The Oligonucleolide primers that designs for PCR

SEQ ID NO：82

The Oligonucleolide primers that designs for PCR

SEQ ID NO：83

The Oligonucleolide primers that designs for PCR

SEQ ID NO：86

The Oligonucleolide primers that designs for PCR

SEQ ID NO：87

The Oligonucleolide primers that designs for PCR

SEQ ID NO：89

The oligonucleotide joint that designs for expression vector establishment

SEQ ID NO：90

The oligonucleotide joint that designs for expression vector establishment

SEQ ID NO：91

The oligonucleotide joint that designs for expression vector establishment

SEQ ID NO：92

The oligonucleotide joint that designs for expression vector establishment

SEQ ID NO：93

The Oligonucleolide primers that designs for PCR

SEQ ID NO：94

The Oligonucleolide primers that designs for PCR

SEQ ID NO：95

The Oligonucleolide primers that designs for PCR

SEQ ID NO：96

The Oligonucleolide primers that designs for PCR

SEQ ID NO：97

The Oligonucleolide primers that designs for PCR

SEQ ID NO：98

The oligonucleotide joint that designs for expression vector establishment

SEQ ID NO：99

The oligonucleotide joint that designs for expression vector establishment

SEQ ID NO：100

The Oligonucleolide primers that designs for PCR

SEQ ID NO：101

The Oligonucleolide primers that designs for PCR

SEQ ID NO：102

The Oligonucleolide primers that designs for PCR

SEQ ID NO：103

The Oligonucleolide primers that designs for PCR

SEQ ID NO：104

The Oligonucleolide primers that designs for PCR

SEQ ID NO：105

The Oligonucleolide primers that designs for PCR

SEQ ID NO：106

The Oligonucleolide primers that designs for PCR

SEQ ID NO：107

The Oligonucleolide primers that designs for PCR

SEQ ID NO：114

The Oligonucleolide primers that designs for PCR

SEQ ID NO：115

The Oligonucleolide primers that designs for PCR

SEQ ID NO：116

The Oligonucleolide primers that designs for PCR

SEQ ID NO：117

The Oligonucleolide primers that designs for PCR

SEQ ID NO：118

The Oligonucleolide primers that designs for PCR

SEQ ID NO：119

The Oligonucleolide primers that designs for PCR

SEQ ID NO：120

The Oligonucleolide primers that designs for PCR

SEQ ID NO：121

The Oligonucleolide primers that designs for PCR

SEQ ID NO：122

The Oligonucleolide primers that designs for PCR

SEQ ID NO：123

The Oligonucleolide primers that designs for PCR

SEQ ID NO：124

The Oligonucleolide primers that designs for PCR

SEQ ID NO：125

The Oligonucleolide primers that designs for PCR

SEQ ID NO：126

The Oligonucleolide primers that designs for PCR

SEQ ID NO：127

The Oligonucleolide primers that designs for PCR

SEQ ID NO：128

The Oligonucleolide primers that designs for PCR

SEQ ID NO：129

The Oligonucleolide primers that designs for PCR

SEQ ID NO：130

The Oligonucleolide primers that designs for PCR

SEQ ID NO：131

The Oligonucleolide primers that designs for PCR

SEQ ID NO：132

The Oligonucleolide primers that designs for PCR

SEQ ID NO：133

The Oligonucleolide primers that designs for PCR

SEQ ID NO：134

The oligonucleotide joint that designs for expression vector establishment

SEQ ID NO：135

The oligonucleotide joint that designs for expression vector establishment

SEQ ID NO：161

The Oligonucleolide primers that designs for PCR

SEQ ID NO：162

The Oligonucleolide primers that designs for PCR

SEQ ID NO：163

The Oligonucleolide primers that designs for PCR

SEQ ID NO：164

The Oligonucleolide primers that designs for PCR

SEQ ID NO：165

The Oligonucleolide primers that designs for PCR

SEQ ID NO：166

The Oligonucleolide primers that designs for PCR

SEQ ID NO：167

The Oligonucleolide primers that designs for PCR

SEQ ID NO：168

The Oligonucleolide primers that designs for PCR

SEQ ID NO：169

The Oligonucleolide primers that designs for PCR

SEQ ID NO：170

The Oligonucleolide primers that designs for PCR

SEQ ID NO：171

The Oligonucleolide primers that designs for PCR

SEQ ID NO：172

The Oligonucleolide primers that designs for PCR

SEQ ID NO：173

The Oligonucleolide primers that designs for PCR

SEQ ID NO：174

The Oligonucleolide primers that designs for PCR

SEQ ID NO：175

The Oligonucleolide primers that designs for PCR

SEQ ID NO：176

The Oligonucleolide primers that designs for PCR

SEQ ID NO：177

The Oligonucleolide primers that designs for PCR

SEQ ID NO：178

The Oligonucleolide primers that designs for PCR

SEQ ID NO：179

The Oligonucleolide primers that designs for PCR

SEQ ID NO：180

The Oligonucleolide primers that designs for PCR

SEQ ID NO：181

The Oligonucleolide primers that designs for PCR

SEQ ID NO：182

The Oligonucleolide primers that designs for PCR

SEQ ID NO：183

The Oligonucleolide primers that designs for PCR

SEQ ID NO：184

The Oligonucleolide primers that designs for PCR

SEQ ID NO：185

The Oligonucleolide primers that designs for PCR

SEQ ID NO：186

The Oligonucleolide primers that designs for dna sequencing

SEQ ID NO：187

The Oligonucleolide primers that designs for dna sequencing

SEQ ID NO：188

The Oligonucleolide primers that designs for dna sequencing

SEQ ID NO：189

The Oligonucleolide primers that designs for dna sequencing

SEQ ID NO：190

The Oligonucleolide primers that designs for PCR

SEQ ID NO：191

The Oligonucleolide primers that designs for PCR

SEQ ID NO：192

The Oligonucleolide primers that designs for PCR

SEQ ID NO：193

The Oligonucleolide primers that designs for PCR

SEQ ID NO：194

The Oligonucleolide primers that designs for PCR

SEQ ID NO：195

The Oligonucleolide primers that designs for PCR

SEQ ID NO：196

The Oligonucleolide primers that designs for PCR

SEQ ID NO：197

The Oligonucleolide primers that designs for PCR

SEQ ID NO：198

The Oligonucleolide primers that designs for PCR

SEQ ID NO：199

The Oligonucleolide primers that designs for PCR

SEQ ID NO；200

The Oligonucleolide primers that designs for PCR

SEQ ID NO：201

The Oligonucleolide primers that designs for PCR

SEQ ID NO：202

The Oligonucleolide primers that designs for PCR

SEQ ID NO：203

The Oligonucleolide primers that designs for PCR

SEQ ID NO：204

The Oligonucleolide primers that designs for PCR

SEQ ID NO：205

The Oligonucleolide primers that designs for PCR

SEQ ID NO：206

The Oligonucleolide primers that designs for PCR

SEQ ID NO：207

The Oligonucleolide primers that designs for PCR

SEQ ID NO：208

The Oligonucleolide primers that designs for PCR

SEQ ID NO：209

The Oligonucleolide primers that designs for PCR

SEQ ID NO：210

The Oligonucleolide primers that designs for PCR

SEQ ID NO：211

The Oligonucleolide primers that designs for PCR

SEQ ID NO：212

The Oligonucleolide primers that designs for PCR

SEQ ID NO：213

The Oligonucleolide primers that designs for PCR

SEQ ID NO：214

The polynucleotide of the design of the aminoacid sequence of coding SEQ ID NO:1

SEQ ID NO：265

The Oligonucleolide primers that designs for PCR

SEQ ID NO：266

The Oligonucleolide primers that designs for PCR

SEQ ID NO：267

The Oligonucleolide primers that designs for PCR

SEQ ID NO：268

The Oligonucleolide primers that designs for PCR

SEQ ID NO：269

The Oligonucleolide primers that designs for PCR

SEQ ID NO：270

The Oligonucleolide primers that designs for PCR

SEQ ID NO：271

The Oligonucleolide primers that designs for PCR

SEQ ID NO：272

The Oligonucleolide primers that designs for PCR

SEQ ID NO：273

The Oligonucleolide primers that designs for PCR

SEQ ID NO：274

The Oligonucleolide primers that designs for PCR

SEQ ID NO：275

The Oligonucleolide primers that designs for PCR

SEQ ID NO：276

The Oligonucleolide primers that designs for dna sequencing

SEQ ID NO：277

The Oligonucleolide primers that designs for dna sequencing

SEQ ID NO：278

The Oligonucleolide primers that designs for PCR

SEQ ID NO：279

The Oligonucleolide primers that designs for PCR

SEQ ID NO：280

The Oligonucleolide primers that designs for PCR

SEQ ID NO：281

The Oligonucleolide primers that designs for dna sequencing

SEQ ID NO：282

The Oligonucleolide primers that designs for PCR

SEQ ID NO：283

The Oligonucleolide primers that designs for PCR

SEQ ID NO：284

The Oligonucleolide primers that designs for PCR

SEQ ID NO：285

The Oligonucleolide primers that designs for PCR

SEQ ID NO：286

The Oligonucleolide primers that designs for PCR

SEQ ID NO：287

The Oligonucleolide primers that designs for PCR

SEQ ID NO：288

The Oligonucleolide primers that designs for dna sequencing

SEQ ID NO：289

The Oligonucleolide primers that designs for PCR

SEQ ID NO：290

The Oligonucleolide primers that designs for PCR

SEQ ID NO：291

The Oligonucleolide primers that designs for PCR

SEQ ID NO：292

The Oligonucleolide primers that designs for PCR

SEQ ID NO：293

The Oligonucleolide primers that designs for PCR

SEQ ID NO：294

The Oligonucleolide primers that designs for PCR

SEQ ID NO：295

The Oligonucleolide primers that designs for PCR

SEQ ID NO：296

The Oligonucleolide primers that designs for PCR

SEQ ID NO：297

The Oligonucleolide primers that designs for PCR

SEQ ID NO：298

The Oligonucleolide primers that designs for PCR

SEQ ID NO：299

The Oligonucleolide primers that designs for PCR

SEQ ID NO：300

The Oligonucleolide primers that designs for dna sequencing

SEQ ID NO：301

The Oligonucleolide primers that designs for PCR

SEQ ID NO：302

The Oligonucleolide primers that designs for PCR

SEQ ID NO：303

The Oligonucleolide primers that designs for PCR

SEQ ID NO：304

The Oligonucleolide primers that designs for PCR

SEQ ID NO：305

The Oligonucleolide primers that designs for PCR

SEQ ID NO：306

The Oligonucleolide primers that designs for PCR

SEQ ID NO：307

The Oligonucleolide primers that designs for PCR

SEQ ID NO：308

The Oligonucleolide primers that designs for dna sequencing

SEQ ID NO：309

The Oligonucleolide primers that designs for PCR

SEQ ID NO：310

The Oligonucleolide primers that designs for PCR

SEQ ID NO：311

The Oligonucleolide primers that designs for PCR

SEQ ID NO：312

The widow who designs for PCR examines former times acid primer

SEQ ID NO：313

The Oligonucleolide primers that designs for PCR

SEQ ID NO：314

The Oligonucleolide primers that designs for PCR

SEQ ID NO：315

The Oligonucleolide primers that designs for dna sequencing

SEQ ID NO：316

The Oligonucleolide primers that designs for PCR

SEQ ID NO：317

The Oligonucleolide primers that designs for PCR

SEQ ID NO：318

The Oligonucleolide primers that designs for PCR

SEQ ID NO：319

The Oligonucleolide primers that designs for PCR

SEQ ID NO：320

The Oligonucleolide primers that designs for PCR

SEQ ID NO：321

The Oligonucleolide primers that designs for PCR

SEQ ID NO：322

The Oligonucleolide primers that designs for dna sequencing

SEQ ID NO：323

The Oligonucleolide primers that designs for PCR

SEQ ID NO：324

The Oligonucleolide primers that designs for PCR

SEQ ID NO：325

The Oligonucleolide primers that designs for PCR

SEQ ID NO：326

The Oligonucleolide primers that designs for PCR

SEQ ID NO：327

The Oligonucleolide primers that designs for PCR

SEQ ID NO：328

The Oligonucleolide primers that designs for PCR

SEQ ID NO：329

The Oligonucleolide primers that designs for PCR

SEQ ID NO：330

The Oligonucleolide primers that designs for PCR

SEQ ID NO：331

The Oligonucleolide primers that designs for PCR

SEQ ID NO：332

The Oligonucleolide primers that designs for PCR

SEQ ID NO：333

The Oligonucleolide primers that designs for PCR

SEQ ID NO：334

The Oligonucleolide primers that designs for PCR

SEQ ID NO：335

The Oligonucleolide primers that designs for PCR

SEQ ID NO：336

The Oligonucleolide primers that designs for PCR

SEQ ID NO：337

The Oligonucleolide primers that designs for PCR

SEQ ID NO：338

The Oligonucleolide primers that designs for PCR

SEQ ID NO：339

The Oligonucleolide primers that designs for PCR

SEQ ID NO：340

The Oligonucleolide primers that designs for PCR

SEQ ID NO：341

The Oligonucleolide primers that designs for PCR

SEQ ID NO：342

The Oligonucleolide primers that designs for PCR

SEQ ID NO：343

The Oligonucleolide primers that designs for PCR

SEQ ID NO：344

The Oligonucleolide primers that designs for PCR

SEQ ID NO：345

The Oligonucleolide primers that designs for PCR

SEQ ID NO：346

The Oligonucleolide primers that designs for PCR

SEQ ID NO：347

The Oligonucleolide primers that designs for PCR

SEQ ID NO：348

The Oligonucleolide primers that designs for PCR

SEQ ID NO：349

The Oligonucleolide primers that designs for PCR

SEQ ID NO：350

The Oligonucleolide primers that designs for PCR

SEQ ID NO：351

The Oligonucleolide primers that designs for PCR

SEQ ID NO：352

The Oligonucleolide primers that designs for PCR

SEQ ID NO：353

The Oligonucleolide primers that designs for PCR

SEQ ID NO：354

The Oligonucleolide primers that designs for PCR

SEQ ID NO：355

The Oligonucleolide primers that designs for PCR

SEQ ID NO：356

The Oligonucleolide primers that designs for PCR

SEQ ID NO：357

The Oligonucleolide primers that designs for PCR

SEQ ID NO：358

The Oligonucleolide primers that designs for PCR

SEQ ID NO：359

The Oligonucleolide primers that designs for PCR

SEQ ID NO：360

The Oligonucleolide primers that designs for PCR

SEQ ID NO：361

The Oligonucleolide primers that designs for PCR

SEQ ID NO：362

The Oligonucleolide primers that designs for PCR

SEQ ID NO：363

The Oligonucleolide primers that designs for PCR

SEQ ID NO：364

The Oligonucleolide primers that designs for PCR

SEQ ID NO：365

The Oligonucleolide primers that designs for PCR

SEQ ID NO：366

The Oligonucleolide primers that designs for PCR

SEQ ID NO：367

The Oligonucleolide primers that designs for PCR

SEQ ID NO：368

The polynucleotide of the design of the aminoacid sequence of coding SEQ ID NO:222

SEQ ID NO：369

The Oligonucleolide primers that designs for PCR

SEQ ID NO：370

The Oligonucleolide primers that designs for PCR

SEQ ID NO：371

The Oligonucleolide primers that designs for PCR

SEQ ID NO：372

The Oligonucleolide primers that designs for PCR

SEQ ID NO：373

The Oligonucleolide primers that designs for PCR

SEQ ID NO：374

The Oligonucleolide primers that designs for PCR

SEQ ID NO：375

The Oligonucleolide primers that designs for PCR

SEQ ID NO：376

The Oligonucleolide primers that designs for PCR

SEQ ID NO：377

The Oligonucleolide primers that designs for PCR

SEQ ID NO：378

The Oligonucleolide primers that designs for PCR

SEQ ID NO：379

The Oligonucleolide primers that designs for PCR

SEQ ID NO：380

The Oligonucleolide primers that designs for PCR

SEQ ID NO：381

The Oligonucleolide primers that designs for PCR

SEQ ID NO：382

The Oligonucleolide primers that designs for PCR

SEQ ID NO：383

The Oligonucleolide primers that designs for PCR

SEQ ID NO：384

The Oligonucleolide primers that designs for PCR

SEQ ID NO：385

The Oligonucleolide primers that designs for PCR

SEQ ID NO：386

The Oligonucleolide primers that designs for PCR

SEQ ID NO：387

The Oligonucleolide primers that designs for PCR

SEQ ID NO：388

The Oligonucleolide primers that designs for PCR

SEQ ID NO：389

The Oligonucleolide primers that designs for PCR

SEQ ID NO：390

The Oligonucleolide primers that designs for PCR

SEQ IDN O：391

The Oligonucleolide primers that designs for PCR

SEQ ID NO：392

The Oligonucleolide primers that designs for PCR

SEQ ID NO：393

The polynucleotide of the design of the aminoacid sequence of coding SEQ ID NO:224

SEQ ID NO：394

The Oligonucleolide primers that designs for PCR

SEQ ID NO：395

The Oligonucleolide primers that designs for PCR

SEQ ID NO：396

The Oligonucleolide primers that designs for PCR

SEQ ID NO：397

The Oligonucleolide primers that designs for PCR

SEQ ID NO：398

The Oligonucleolide primers that designs for PCR

SEQ ID NO：399

The Oligonucleolide primers that designs for PCR

SEQ ID NO：400

The Oligonucleolide primers that designs for PCR

SEQ ID NO：401

The Oligonucleolide primers that designs for PCR

SEQ ID NO：402

The oligonucleotide joint that designs for expression vector establishment

SEQ ID NO：403

The oligonucleotide joint that designs for expression vector establishment

The PC040425-sequence table

Sequence table

＜110〉Sumitomo Chemical Company Ltd

＜120〉metabolic protein for weed-control agent, its gene and application thereof

<130>561096

<150>JP 2001/321307

<151>2001-10-19

<150>JP 2002/167239

<151>2002-06-07

<160>403

<210>1

<211>408

<212>PRT

＜213〉abortion within the first month of pregnancy look streptomycete (Streptmyces phaeochromogenes) IFO 12898

<400>1

<210>2

<211>401

<212>PRT

＜213〉Ta Shi saccharopolyspora strain (Saccharopolyspora taberi) JCM 9383t

<400>2

<210>3

<211>395

<212>PRT

＜213〉brick-red streptomycete (Streptmyces testaceus) ATCC 21469

<400>3

<210>4

<211>410

<212>PRT

<213>Streptmyces carbophi lus SANK 62585

<400>4

<210>5

<211>406

<212>PRT

＜213〉light gray streptomycete (Streptmyces griseolus) ATCC 11796

<400>5

<210>6

<211>1227

<212>DNA

<220>

<221>CDS

<222>(1)..(1227)

<400>6

<210>7

<211>1206

<212>DNA

＜213〉Ta Shi saccharopolyspora strain (Saccharopolyspora taberi) JCM 9383t

<220>

<221>CDS

<222>(1)..(1206)

<400>7

<210>8

<211>1188

<212>DNA

＜213〉brick-red streptomycete (Streptmyces testaceus) ATCC 21469

<220>

<221>CDS

<222>(1)..(1188)

<400>8

<210>9

<211>1439

<212>DNA

<220>

<221>CDS

<222>(1)..(1227)

<220>

<221>CDS

<222>(1239)..(1439)

<400>9

<210>10

<211>1415

<212>DNA

＜213〉Ta Shi saccharopolyspora strain (Saccharopolyspora taberi) JCM 9383t

<220>

<221>CDS

<222>(1)..(1206)

<220>

<221>CDS

<222>(1218)..(1415)

<400>10

<210>11

<211>1418

<212>DNA

＜213〉brick-red streptomycete (Streptmyces testaceus) ATCC 21469

<220>

<221>CDS

<222>(1)..(1188)

<220>

<221>CDS

<222>(1224)..(1418)

<400>11

<210>12

<211>66

<212>PRT

<400>12

<210>13

<211>65

<212>PRT

＜213〉Ta Shi saccharopolyspora strain (Saccharopolyspora taberi) JCM 9383t

<400>13

<210>14

<211>64

<212>PRT

＜213〉brick-red streptomycete (Streptmyces testaceus) ATCC 21469

<400>14

<210>15

<211>201

<212>DNA

<220>

<221>CDS

<222>(1)..(201)

<400>15

<210>16

<211>198

<212>DNA

＜213〉Ta Shi saccharopolyspora strain (Saccharopolyspora taberi) JCM 9383t

<220>

<221>CDS

<222>(1)..(198)

<400>16

<210>17

<211>195

<212>DNA

＜213〉brick-red streptomycete (Streptmyces testaceus) ATCC 21469

<220>

<221>CDS

<222>(1)..(195)

<400>17

<210>18

<211>23

<212>PRT

<400>18

<210>19

<211>7

<212>PRT

<400>19

<210>20

<211>19

<212>PRT

＜213〉Ta Shi saccharopolyspora strain (Saccharopolyspora taberi) JCM 9383T

<400>20

<210>21

<211>8

<212>PRT

＜213〉Ta Shi saccharopolyspora strain (Saccharopolyspora taberi) JCM 9383T

<400>21

<210>22

<211>19

<212>PRT

＜213〉light gray streptomycete (Streptomyces griseolus) ATCC11796

<400>22

<210>23

<211>15

<212>PRT

＜213〉light gray streptomycete (Streptomyces griseolus) ATCC11796

<400>23

<210>24

<211>15

<212>PRT

＜213〉light gray streptomycete (Streptomyces griseolus) ATCC11796

<400>24

<210>25

<211>7

<212>PRT

＜213〉light gray streptomycete (Streptomyces griseolus) ATCC11796

<400>25

<210>26

<211>11

<212>PRT

＜213〉light gray streptomycete (Streptomyces griseolus) ATCC11796

<400>26

<210>27

<211>17

<212>PRT

＜213〉light gray streptomycete (Streptomyces griseolus) ATCC11796

<400>27

<210>28

<211>9

<212>PRT

＜213〉light gray streptomycete (Streptomyces griseolus) ATCC11796

<400>28

<210>29

<211>14

<212>PRT

＜213〉light gray streptomycete (Streptomyces griseolus) ATCC11796

<400>29

<210>30

<211>12

<212>PRT

＜213〉light gray streptomycete (Streptomyces griseolus) ATCC11796

<400>30

<210>31

<211>11

<212>PRT

＜213〉light gray streptomycete (Streptomyces griseolus) ATCC11796

<400>31

<210>32

<211>14

<212>PRT

＜213〉light gray streptomycete (Streptomyces griseolus) ATCC11796

<400>32

<210>33

<211>15

<212>PRT

＜213〉light gray streptomycete (Streptomyces griseolus) ATCC11796

<400>33

<210>34

<211>13

<212>PRT

＜213〉light gray streptomycete (Streptomyces griseolus) ATCC11796

<400>34

<210>35

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>35

<210>36

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>36

<210>37

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>37

<210>38

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>38

<210>39

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>39

<210>40

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>40

<210>41

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>41

<210>42

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>42

<210>43

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>43

<210>44

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>44

<210>45

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>45

<210>46

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>46

<210>47

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>47

<210>48

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>48

<210>49

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>49

<210>50

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>50

<210>51

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>51

<210>52

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>52

<210>53

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>53

<210>54

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>54

<210>55

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>55

<210>56

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>56

<210>57

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>57

<210>58

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>58

<210>59

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>59

<210>60

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>60

<210>61

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>61

<210>62

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>62

<210>63

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>63

<210>64

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>64

<210>65

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>65

<210>66

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>66

<210>67

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>67

<210>68

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>68

<210>69

<211>1418

<212>DNA

＜213〉brick-red streptomycete (Streptmyces testaceus) ATCC 21469

<220>

<221>CDS

<222>(1)..(1188)

<220>

<221>CDS

<222>(1224)..(1418)

<400>69

<210>70

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>70

<210>71

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>71

<210>72

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>72

<210>73

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>73

<210>74

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>74

<210>75

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>75

<210>76

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>76

<210>77

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>77

<210>78

<211>1233

<212>DNA

<213>Streptmyces carbophilus SANK 62585

<220>

<221>CDS

<222>(1)..(1233)

<400>78

<210>79

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>79

<210>80

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>80

<210>81

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>81

<210>82

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>82

<210>83

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>83

<210>84

<211>1221

<212>DNA

＜213〉light gray streptomycete (Streptmyces griseolus) ATCC 11796

<220>

<221>CDS

<222>(1)..(1221)

<400>84

<210>85

<211>1451

<212>DNA

＜213〉light gray streptomycete (Streptmyces griseolus) ATCC 11796

<220>

<221>CDS

<222>(1)..(1221)

<220>

<221>CDS

<222>(1242)..(1451)

<400>85

<210>86

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>86

<210>87

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>87

<210>88

<211>248

<212>DNA

<213>Glycine max(L.)Merrill

<220>

<221>CDS

<222>(20)..(220)

<400>88

<210>89

<211>9

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide joint that designs for expression vector establishment

<400>89

<210>90

<211>13

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide joint that designs for expression vector establishment

<400>90

<210>91

<211>13

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide joint that designs for expression vector establishment

<400>91

<210>92

<211>11

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide joint that designs for expression vector establishment

<400>92

<210>93

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>93

<210>94

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>94

<210>95

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>95

<210>96

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>96

<210>97

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>97

<210>98

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide joint that designs for expression vector establishment

<400>98

<210>99

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide joint that designs for expression vector establishment

<400>99

<210>100

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>100

<210>101

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>101

<210>102

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>102

<210>103

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>103

<210>104

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>104

<210>105

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>105

<210>106

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>106

<210>107

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>107

<210>108

<211>411

<212>PRT

＜213〉do not produce look streptomycete (Streptomyces achromogenes) IFO 12735

<400>108

<210>109

<211>1236

<212>DNA

<220>

<221>CDS

<222>(1)..(1236)

<400>109

<210>110

<211>1454

<212>DNA

<220>

<221>CDS

<222>(1)..(1236)

<220>

<221>CDS

<222>(1263)..(1454)

<400>110

<210>111

<211>63

<212>PRT

<400>111

<210>112

<211>192

<212>DNA

<220>

<221>CDS

<222>(1)..(192)

<400>112

<210>113

<211>20

<212>PRT

<400>113

<210>114

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>114

<210>115

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>115

<210>116

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>116

<210>117

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>117

<210>118

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>118

<210>119

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>119

<210>120

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>120

<210>121

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>121

<210>122

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>122

<210>123

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>123

<210>124

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>124

<210>125

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>125

<210>126

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>126

<210>127

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>127

<210>128

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>128

<210>129

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>129

<210>130

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>130

<210>131

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>131

<210>132

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>132

<210>133

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>133

<210>134

<211>72

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide joint that designs for expression vector establishment

<400>134

<210>135

<211>68

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide joint that designs for expression vector establishment

<400>135

<210>136

<211>402

<212>PRT

＜213〉hot lightskyblue streptomycete (Streptmyces thermocoerulescens) IFO 14273t

<400>136

<210>137

<211>409

<212>PRT

＜213〉pelletizing produces look streptomycete (Streptmyces glomerochromogenes) IFO 13673T

<400>137

<210>138

<211>409

<212>PRT

＜213〉olive produces look streptomycete (Streptmyces olivochromogenes) IFO 12444

<400>138

<210>139

<211>1230

<212>DNA

＜213〉black walnut streptomycete (Streptmyces nogalater) IFO 13445

<220>

<221>CDS

<222>(1)..(1230)

<400>139

<210>140

<211>1278

<212>DNA

＜213〉Tianjin island chain mould (Streptmyces tsusimaensis) IFO 13782T

<220>

<221>CDS

<222>(1)..(1278)

<400>140

<210>141

<211>1209

<212>DNA

<220>

<221>CDS

<222>(1)..(1209)

<400>141

<210>142

<211>1230

<212>DNA

<220>

<221>CDS

<222>(1)..(1230)

<400>142

<210>143

<211>1230

<212>DNA

<220>

<221>CDS

<222>(1)..(1230)

<400>143

<210>144

<211>1449

<212>DNA

＜213〉black walnut streptomycete (Streptmyces nogalater) IFO 13445

<220>

<221>CDS

<222>(1)..(1230)

<220>

<221>CDS

<222>(1243)..(1449)

<400>144

<210>145

<211>1480

<212>DNA

＜213〉Tianjin island chain mould (Streptmyces tsusimaensis) IFO 13782T

<220>

<221>CDS

<222>(1)..(1278)

<220>

<221>CDS

<222>(1284)..(1480)

<400>145

<210>146

<211>1473

<212>DNA

＜213〉hot lightskyblue streptomycete (Streptomyces thermocoerulescens) IFO 14273t

<220>

<221>CDS

<222>(1)..(1209)

<220>

<221>CDS

<222>(1222)..(1473)

<400>146

<210>147

<211>1449

<212>DNA

<220>

<221>CDS

<222>(1)..(1230)

<220>

<221>CDS

<222>(1243)..(1449)

<400>147

<210>148

<211>1449

<212>DNA

<220>

<221>CDS

<222>(1)..(1230)

<220>

<221>CDS

<222>(1243)..(1449)

<400>148

<210>149

<211>68

<212>PRT

＜213〉black walnut streptomycete (Streptmyces nogalater) IFO 13445

<400>149

<210>150

<211>65

<212>PRT

＜213〉Tianjin island chain mould (Streptmyces tsusimaensis) IFO 13782T

<400>150

<210>151

<211>83

<212>PRT

<400>151

<210>152

<211>68

<212>PRT

<400>152

<210>153

<211>68

<212>PRT

<400>153

<210>154

<211>207

<212>DNA

＜213〉black walnut streptomycete (Streptmyces nogalater) IFO 13445

<220>

<221>CDS

<222>(1)..(207)

<400>154

<210>155

<211>198

<212>DNA

＜213〉Tianjin island chain mould (Streptmyces tsusimaensis) IFO 13782T

<220>

<221>CDS

<222>(1)..(198)

<400>155

<210>156

<211>252

<212>DNA

<220>

<221>CDS

<222>(1)..(252)

<400>156

<210>157

<211>207

<212>DNA

<220>

<221>CDS

<222>(1)..(207)

<400>157

<210>158

<211>207

<212>DNA

<220>

<221>CDS

<222>(1)..(207)

<400>158

<210>159

<211>409

<212>PRT

＜213〉black walnut streptomycete (Streptmyces nogalater) IFO 13445

<400>159

<210>160

<211>425

<212>PRT

＜213〉Tianjin island chain mould (Streptmyces tsusimaensis) IFO 13782T

<400>160

<210>161

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>161

<210>162

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>162

<210>163

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>163

<210>164

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>164

<210>165

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>165

<210>166

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>166

<210>167

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>167

<210>168

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>168

<210>169

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>169

<210>170

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>170

<210>171

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>171

<210>172

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>172

<210>173

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>173

<210>174

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>174

<210>175

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>175

<210>176

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>176

<210>177

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>177

<210>178

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>178

<210>179

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>179

<210>180

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>180

<210>181

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>181

<210>182

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>182

<210>183

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>183

<210>184

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>184

<210>185

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>185

<210>186

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for dna sequencing

<400>186

<210>187

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for dna sequencing

<400>187

<210>188

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for dna sequencing

<400>188

<210>189

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for dna sequencing

<400>189

<210>190

<211>78

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>190

<210>191

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>191

<210>192

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>192

<210>193

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>193

<210>194

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>194

<210>195

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>195

<210>196

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>196

<210>197

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>197

<210>198

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>198

<210>199

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>199

<210>200

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>200

<210>201

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>201

<210>202

<211>79

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>202

<210>203

<211>80

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>203

<210>204

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>204

<210>205

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>205

<210>206

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>206

<210>207

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>207

<210>208

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>208

<210>209

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>209

<210>210

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>210

<210>211

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>211

<210>212

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>212

<210>213

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>213

<210>214

<211>1227

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)..(1227)

＜223〉polynucleotide of the aminoacid sequence of She Ji coding SEQ ID No.1

<400>214

<210>215

<211>416

<212>PRT

＜213〉decorative chains mould (Streptomyces ornatus) IFO 13069t

<400>215

<210>216

<211>416

<212>PRT

＜213〉streptomyces griseus (Streptomyces griseus) ATCC 10137

<400>216

<210>217

<211>409

<212>PRT

<400>217

<210>218

<211>416

<212>PRT

＜213〉streptomyces griseus (Streptomyces griseus) IFO 13849T

<400>218

<210>219

<211>405

<212>PRT

＜213〉wool streptomycete (Streptomyces lanatus) IFO 12787T

<400>219

<210>220

<211>414

<212>PRT

＜213〉three damp streptomycetes (Streptomyces misawanensis) IFO 13855T

<400>220

<210>221

<211>409

<212>PRT

＜213〉pale asphyxia streptomycete (Streptomyces pallidus) IFO 13434T

<400>221

<210>222

<211>398

<212>PRT

＜213〉rose streptomycete (Streptomyces roseorubens) the IFO 13682T that reddens

<400>222

<210>223

<211>414

<212>PRT

＜213〉ground, Shandong streptomycete (Streptomyces rutgersensis) IFO 15875T

<400>223

<210>224

<211>398

<212>PRT

＜213〉Regensburg streptomycete (Streptomyces steffisburgensis) IFO 13446T

<400>224

<210>225

<211>1251

<212>DNA

＜213〉decorative chains mould (Streptomyces ornatus) IFO 13069t

<220>

<221>CDS

<222>(1)..(1251)

<400>225

<210>226

<211>1251

<212>DNA

＜213〉streptomyces griseus (Streptomyces griseus) ATCC 10137

<220>

<221>CDS

<222>(1)..(1251)

<400>226

<210>227

<211>1230

<212>DNA

<220>

<221>CDS

<222>(1)..(1230)

<400>227

<210>228

<211>1251

<212>DNA

＜213〉streptomyces griseus (Streptomyces griseus) IFO 13849T

<220>

<221>CDS

<222>(1)..(1251)

<400>228

<210>229

<211>1218

<212>DNA

＜213〉wool streptomycete (Streptomyces lanatus) IFO 12787T

<220>

<221>CDS

<222>(1)..(1218)

<400>229

<210>230

<211>1245

<212>DNA

＜213〉three damp streptomycetes (Streptomyces misawanensis) IFO 13855T

<220>

<221>CDS

<222>(1)..(1245)

<400>230

<210>231

<211>1230

<212>DNA

＜213〉pale asphyxia streptomycete (Streptomyces pallidus) IFO 13434T

<220>

<221>CDS

<222>(1)..(1230)

<400>231

<210>232

<211>1197

<212>DNA

<220>

<221>CDS

<222>(1)..(1197)

<400>232

<210>233

<211>1245

<212>DNA

＜213〉ground, Shandong streptomycete (Streptomyces rutgersensis) IFO 15875T

<220>

<221>CDS

<222>(1)..(1245)

<400>233

<210>234

<211>1197

<212>DNA

＜213〉Regensburg streptomycete (Streptomyces steffisburgensis) IFO 13446T

<220>

<221>CDS

<222>(1)..(1197)

<400>234

<210>235

<211>1454

<212>DNA

＜213〉decorative chains mould (Streptomyces ornatus) IFO 13069t

<220>

<221>CDS

<222>(1)..(1251)

<220>

<221>CDS

<222>(1257)..(1454)

<400>235

<210>236

<211>1454

<212>DNA

＜213〉streptomyces griseus (Streptomyces griseus) ATCC 10137

<220>

<221>CDS

<222>(1)..(1251)

<220>

<221>CDS

<222>(1257)..(1454)

<400>236

<210>237

<211>1449

<212>DNA

<220>

<221>CDS

<222>(1)..(1230)

<220>

<221>CDS

<222>(1243)..(1449)

<400>237

<210>238

<211>1454

<212>DNA

＜213〉streptomyces griseus (Streptomyces griseus) IFO 13849T

<220>

<221>CDS

<222>(1)..(1251)

<220>

<221>CDS

<222>(1299)..(1454)

<400>238

<210>239

<211>1461

<212>DNA

＜213〉wool streptomycete (Streptomyces lanatus) IFO 12787T

<220>

<221>CDS

<222>(1)..(1218)

<220>

<221>CDS

<222>(1231)..(1461)

<400>239

<210>240

<211>1458

<212>DNA

＜213〉three damp streptomycetes (Streptomyces misawanensis) IFO 13855T

<220>

<221>CDS

<222>(1)..(1245)

<220>

<221>CDS

<222>(1258)..(1458)

<400>240

<210>241

<211>1448

<212>DNA

＜213〉pale asphyxia streptomycete (Streptomyces pallidus) IFO 13434T

<220>

<221>CDS

<222>(1)..(1230)

<220>

<221>CDS

<222>(1254)..(1448)

<400>241

<210>242

<211>1411

<212>DNA

<220>

<221>CDS

<222>(1)..(1197)

<220>

<221>CDS

<222>(1211)..(1411)

<400>242

<210>243

<211>1466

<212>DNA

＜213〉ground, Shandong streptomycete (Streptomyces rutgersensis) IFO 15875T

<220>

<221>CDS

<222>(1)..(1245)

<220>

<221>CDS

<222>(1269)..(1466)

<400>243

<210>244

<211>1411

<212>DNA

＜213〉Regensburg streptomycete (Streptomyces steffisburgensis) IFO 13446T

<220>

<221>CDS

<222>(1)..(1197)

<220>

<221>CDS

<222>(1211)..(1411)

<400>244

<210>245

<211>65

<212>PRT

＜213〉decorative chains mould (Streptomyces ornatus) IFO 13069t

<400>245

<210>246

<211>65

<212>PRT

＜213〉streptomyces griseus (Streptomyces griseus) ATCC 10137

<400>246

<210>247

<211>68

<212>PRT

<400>247

<210>248

<211>51

<212>PRT

＜213〉streptomyces griseus (Streptomyces griseus) IFO 13849T

<400>248

<210>249

<211>76

<212>PRT

＜213〉wool streptomycete (Streptomyces lanatus) IFO 12787T

<400>249

<210>250

<211>66

<212>PRT

＜213〉three damp streptomycetes (Streptomyces misawanensis) IFO 13855T

<400>250

<210>251

<211>64

<212>PRT

＜213〉pale asphyxia streptomycete (Streptomyces pallidus) IFO 13434T

<400>251

<210>252

<211>66

<212>PRT

<400>252

<210>253

<211>65

<212>PRT

＜213〉ground, Shandong streptomycete (Streptomyces rutgersensis) IFO 15875T

<400>253

<210>254

<211>66

<212>PRT

＜213〉Regensburg streptomycete (Streptomyces steffisburgensis) IFO 13446T

<400>254

<210>255

<211>198

<212>DNA

＜213〉decorative chains mould (Streptomyces ornatus) IFO 13069t

<220>

<221>CDS

<222>(1)..(198)

<400>255

<210>256

<211>198

<212>DNA

＜213〉streptomyces griseus (Streptomyces griseus) ATCC 10137

<220>

<221>CDS

<222>(1)..(198)

<400>256

<210>257

<211>207

<212>DNA

<220>

<221>CDS

<222>(1)..(207)

<400>257

<210>258

<211>156

<212>DNA

＜213〉streptomyces griseus (Streptomyces griseus) IFO 13849T

<220>

<221>CDS

<222>(1)..(156)

<400>258

<210>259

<211>231

<212>DNA

＜213〉wool streptomycete (Streptomyces lanatus) IFO 12787T

<220>

<221>CDS

<222>(1)..(231)

<400>259

<210>260

<211>201

<212>DNA

＜213〉three damp streptomycetes (Streptomyces misawanensis) IFO 13855T

<220>

<221>CDS

<222>(1)..(201)

<400>260

<210>261

<211>195

<212>DNA

＜213〉pale asphyxia streptomycete (Streptomyces pallidus) IFO 13434T

<220>

<221>CDS

<222>(1)..(195)

<400>261

<210>262

<211>201

<212>DNA

<220>

<221>CDS

<222>(1)..(201)

<400>262

<210>263

<211>198

<212>DNA

＜213〉ground, Shandong streptomycete (Streptomyces rutgersensis) IFO 15875T

<220>

<221>CDS

<222>(1)..(198)

<400>263

<210>264

<211>201

<212>DNA

＜213〉Regensburg streptomycete (Streptomyces steffisburgensis) IFO 13446T

<220>

<221>CDS

<222>(1)..(201)

<400>264

<210>265

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>265

<210>266

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>266

<210>267

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>267

<210>268

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>268

<210>269

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>269

<210>270

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>270

<210>271

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>271

<210>272

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>272

<210>273

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>273

<210>274

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>274

<210>275

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>275

<210>276

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for dna sequencing

<400>276

<210>277

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for dna sequencing

<400>277

<210>278

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>278

<210>279

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>279

<210>280

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>280

<210>281

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for dna sequencing

<400>281

<210>282

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>282

<210>283

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>283

<210>284

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>284

<210>285

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>285

<210>286

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>286

<210>287

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>287

<210>288

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for dna sequencing

<400>288

<210>289

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>289

<210>290

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>290

<210>291

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>291

<210>292

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>292

<210>293

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>293

<210>294

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>294

<210>295

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>295

<210>296

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>296

<210>297

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>297

<210>298

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>298

<210>299

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>299

<210>300

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for dna sequencing

<400>300

<210>301

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>301

<210>302

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>302

<210>303

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>303

<210>304

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>304

<210>305

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>305

<210>306

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>306

<210>307

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>307

<210>308

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for dna sequencing

<400>308

<210>309

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>309

<210>310

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>310

<210>311

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>311

<210>312

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>312

<210>313

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>313

<210>314

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>314

<210>315

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for dna sequencing

<400>315

<210>316

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>316

<210>317

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>317

<210>318

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>318

<210>319

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>319

<210>320

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>320

<210>321

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>321

<210>322

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for dna sequencing

<400>322

<210>323

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>323

<210>324

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>324

<210>325

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>325

<210>326

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>326

<210>327

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>327

<210>328

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>328

<210>329

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>329

<210>330

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>330

<210>331

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>331

<210>332

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>332

<210>333

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>333

<210>334

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>334

<210>335

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>335

<210>336

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>336

<210>337

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>337

<210>338

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>338

<210>339

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>339

<210>340

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>340

<210>341

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>341

<210>342

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>342

<210>343

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>343

<210>344

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>344

<210>345

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>345

<210>346

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>346

<210>347

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>347

<210>348

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>348

<210>349

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>349

<210>350

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>350

<210>351

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>351

<210>352

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>352

<210>353

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>353

<210>354

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>354

<210>355

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>355

<210>356

<211>56

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>356

<210>357

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>357

<210>358

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>358

<210>359

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>359

<210>360

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>360

<210>361

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>361

<210>362

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>362

<210>363

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>363

<210>364

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>364

<210>365

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>365

<210>366

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>366

<210>367

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>367

<210>368

<211>1197

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)..(1197)

＜223〉polynucleotide of the aminoacid sequence of She Ji coding SEQ ID No.222

<400>368

<210>369

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>369

<210>370

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>370

<210>371

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>371

<210>372

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>372

<210>373

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>373

<210>374

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>374

<210>375

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>375

<210>376

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>376

<210>377

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>377

<210>378

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>378

<210>379

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>379

<210>380

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>380

<210>381

<211>56

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>381

<210>382

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>382

<210>383

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>383

<210>384

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>384

<210>385

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>385

<210>386

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>386

<210>387

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for pCR

<400>387

<210>388

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>388

<210>389

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>389

<210>390

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>390

<210>391

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>391

<210>392

<211>70

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>392

<210>393

<211>1197

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)..(1197)

＜223〉polynucleotide of the aminoacid sequence of She Ji coding SEQ ID No.224

<400>393

<210>394

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>394

<210>395

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>395

<210>396

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>396

<210>397

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>397

<210>398

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>398

<210>399

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>399

<210>400

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>400

<210>401

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers that designs for PCR

<400>401

<210>402

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide joint that designs for expression vector establishment

<400>402

<210>403

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide joint that designs for expression vector establishment

<400>403

Claims

1. the DNA of coded protein, described protein are selected from the group that following substances is formed:

A kind of protein, its aminoacid sequence is shown in SEQ ID NO:224; With

A kind of protein, aminoacid sequence shown in its aminoacid sequence and the SEQ ID NO:224 has at least 90% sequence identity, and wherein said protein has the ability that formula (II) compound is converted into formula (III) compound in the presence of the electron transport system that comprises electron donor:

2. according to the DNA of claim 1, wherein said proteinic aminoacid sequence is shown in SEQ IDNO:222 or SEQ ID NO:224.

3. according to the DNA of claim 1, wherein said proteinic aminoacid sequence nucleotide sequence coded by shown in SEQ IDNO:232 or the SEQ ID NO:234.

4. according to the DNA of claim 1, it comprises the nucleotide sequence of the aminoacid sequence of code for said proteins, and wherein to use be that codon uses in ± 4% the scope and the GC content of described nucleotide sequence is at least 40% and at the most 60% in from the gene of the host cell species of introducing described DNA to the codon in described nucleotide sequence.

5. according to the DNA of claim 4, wherein said nucleotides sequence is listed among the SEQ ID NO:368 to be represented.

6. according to the DNA of claim 4, wherein said nucleotides sequence is listed among the SEQ ID NO:393 to be represented.

7. DNA, the DNA that wherein has a nucleotide sequence of coding born of the same parents inner cell organ encoding transport signals sequence is connected the upstream according to the DNA of claim 1 in frame.

8. a DNA wherein is operably connected according to the DNA of claim 1 and the promotor that works in host cell.

9. comprise carrier according to the DNA of claim 1.

10. method of producing carrier, it comprises will insert the step of the carrier that can duplicate according to the DNA of claim 1 in host cell.

11. a transformant wherein will be introduced host cell according to the DNA of claim 1.

12. according to the transformant of claim 11, wherein said host cell is microorganism cells or vegetable cell.

13. a method of producing transformant, it comprises and will introduce the step of host cell according to the DNA of claim 1.

14. a production has the method for protein that formula (II) compound is converted into the ability of formula (III) compound, described method comprises cultivation according to the transformant of claim 11 and the described proteinic step of recovery generation.

15. have application in the protein of the ability that formula (II) compound is converted into formula (III) compound in production according to the DNA of claim 1.

16. a method that gives the plant herbicide resistance, described method comprise according to the DNA introduced plant cell of claim 1 with the step of expressing therein.

17. one kind is detected coding and has the method for protein DNA that formula (II) compound is converted into the ability of formula (III) compound, described method comprise detection in hybridization with the step of the DNA of probe hybridization, described hybridization uses DNA according to claim 1 as probe.

18. one kind obtains coding and has the method for protein DNA that formula (II) compound is converted into the ability of formula (III) compound, described method comprises the step that reclaims the DNA that detects by the method according to claim 17.

19. a screening has the method for the cell of a kind of protein DNA of coding, described protein has the ability that formula (II) compound is converted into formula (III) compound, and described method comprises by detect the step of described DNA from the test cell according to the method for claim 17.

20. a protein, described protein are selected from the group that following substances is formed:

A kind of protein, its aminoacid sequence is shown in SEQ ID NO:224; With

21. according to the protein of claim 20, wherein said proteinic aminoacid sequence is shown in SEQID NO:222 or SEQ ID NO:224.

22. an identification is according to the proteinic antibody of claim 20.

23. a detection is according to the method for protein of claim 1, described method comprises:

(1) with sample material and the step that contact of the described proteinic antibody of identification with

(2) detect the step of complex body that produce by described contact, described protein and described antibody.

24. analyze or detection kit for one kind, it comprises the antibody according to claim 22.

25. a DNA who comprises nucleotide sequence, described Nucleotide is selected from the group of being made up of following substances:

The nucleotide sequence of in SEQ ID NO:242, representing;

With

The nucleotide sequence of in SEQ ID NO:244, representing.

26. comprise carrier according to the DNA of claim 25.

27. a transformant wherein will be introduced host cell according to the DNA of claim 25.

28. according to the transformant of claim 27, wherein said host cell is microorganism cells or vegetable cell.

29. a method of producing transformant, it comprises and will introduce the step of host cell according to the DNA of claim 25.

30. a production has the method for protein that formula (II) compound is converted into the ability of formula (III) compound, described method comprises cultivation according to the transformant of claim 27 and the described proteinic step of recovery generation.

31. a control method for weed, it comprises compound administration in the step of expressing according to the proteinic cultivation of plants zone of claim 20,

Wherein said compound is the compound of formula (I):

Wherein in G-1 to G-9,

X represents Sauerstoffatom or sulphur atom;

Y represents Sauerstoffatom or sulphur atom;

R ¹Expression hydrogen atom or halogen atom;

R ²The expression hydrogen atom, C ₁-C ₈Alkyl, C ₁-C ₈Haloalkyl, halogen atom, hydroxyl ,-OR ⁹Group ,-SH group ,-S (O) pR ⁹Group ,-COR ⁹Group ,-CO ₂R ⁹Group ,-C (O) SR ⁹Group ,-C (O) NR ¹¹R ¹²Group ,-CONH ₂Group ,-CHO group ,-CR ⁹=NOR ¹⁸Group ,-CH=CR ¹⁹CO ₂R ⁹Group ,-CH ₂CHR ¹⁹CO ₂R ⁹Group ,-CO ₂N=CR ¹³R ¹⁴Group, nitro, cyano group ,-NHSO ₂R ¹⁵Group ,-NHSO ₂NHR ¹⁵Group ,-NR ⁹R ²⁰Group ,-NH ₂Group or phenyl, they can be by one or more C that can be identical or different ₁-C ₄Alkyl replaces;

P represents 0,1 or 2;

R ³Expression C ₁-C ₂Alkyl, C ₁-C ₂Haloalkyl ,-OCH ₃Group ,-SCH ₃Group ,-OCHF ₂Group, halogen atom, cyano group, nitro or C ₁-C ₃Alkoxyl group, it is used on the phenyl ring and can be replaced by the phenyl that at least one substituting group replaces, and described substituting group is selected from halogen atom, C ₁-C ₃Alkyl, C ₁-C ₃Haloalkyl, OR ²⁸Group, NR ¹¹R ²⁸Group, SR ²⁸Group, cyano group, CO ₂R ²⁸Group and nitro;

R ⁴The expression hydrogen atom, C ₁-C ₃Alkyl or C ₁-C ₃Haloalkyl;

R ⁸The expression hydrogen atom, C ₁-C ₈Alkyl, C ₃-C ₈Cycloalkyl, C ₃-C ₈Thiazolinyl, C ₃-C ₈Alkynyl, C ₁-C ₈Haloalkyl, C ₂-C ₈Alkoxyalkyl, C ₃-C ₈Alkoxy alkoxy alkyl, C ₃-C ₈The halo alkynyl, C ₃-C ₅Haloalkenyl group, C ₁-C ₈Alkyl sulphonyl, C ₁-C ₈Halogenated alkyl sulfonyl, C ₃-C ₈Alkoxy carbonyl alkyl ,-S (O) ₂NH (C ₁-C ₈Alkyl) group ,-C (O) R ²³Group or can be by R on phenyl ring ²⁴The benzyl that replaces;

R ⁹Expression C ₁-C ₈Alkyl, C ₃-C ₈Cycloalkyl, C ₃-C ₈Thiazolinyl, C ₃-C ₈Alkynyl, C ₁-C ₈Haloalkyl, C ₂-C ₈Alkoxyalkyl, C ₂-C ₈Alkyl-thio-alkyl, C ₂-C ₈The alkyl sulphinyl alkyl, C ₂-C ₈The alkyl sulphonyl alkyl, C ₄-C ₈Alkoxy alkoxy alkyl, C ₄-C ₈Cycloalkylalkyl, C ₄-C ₈The cycloalkyloxy alkyl, C ₄-C ₈Thiazolinyl oxygen base alkyl, C ₄-C ₈Alkynyloxy base alkyl, C ₃-C ₈Halogenated alkoxy alkyl, C ₄-C ₈Haloalkenyl group oxygen base alkyl, C ₄-C ₈Halo alkynyloxy base alkyl, C ₄-C ₈The cycloalkyl alkylthio, C ₄-C ₈The thiazolinyl alkylthio, C ₄-C ₈The alkynyl alkylthio, the C that phenoxy group replaces ₁-C ₄Alkyl, described phenoxy group can be replaced by at least a substituting group on ring, and described substituting group is selected from halogen atom, C ₁-C ₃Alkyl and C ₁-C ₃Haloalkyl, the C that benzyloxy replaces ₁-C ₄Alkyl, described benzyloxy can be replaced by at least a substituting group on ring, and described substituting group is selected from halogen atom, C ₁-C ₃Alkyl and C ₁-C ₃Haloalkyl, C ₄-C ₈The trialkylsyrylalkyl group, C ₂-C ₈Qing Wanji, C ₃-C ₈Halogenated cycloalkyl, C ₃-C ₈Haloalkenyl group, C ₅-C ₈The alkoxyl group thiazolinyl, C ₅-C ₈The halogenated alkoxy thiazolinyl, C ₅-C ₈The alkylthio thiazolinyl, C ₃-C ₈The halo alkynyl, C ₅-C ₈The alkoxyl group alkynyl, C ₅-C ₈The halogenated alkoxy alkynyl, C ₅-C ₈The alkylthio alkynyl, C ₂-C ₈Alkyl-carbonyl can be with at least a halogen atom that is selected from, C on ring ₁-C ₃Alkyl, C ₁-C ₃Haloalkyl ,-OR ²⁸Group ,-NR ¹¹R ²⁸Group ,-SR ²⁸Group, cyano group ,-CO ₂R ²⁸The benzyl that the substituting group of group and nitro replaces ,-CR ¹⁶R ¹⁷COR ¹⁰Group ,-CR ¹⁶R ¹⁷CO ₂R ²⁰Group ,-CR ¹⁶R ¹⁷P (O) (OR ¹⁰) ₂Group ,-CR ¹⁶R ¹⁷P (S) (OR ¹⁰) ₂Group ,-CR ¹⁶R ¹⁷C (O) NR ¹¹R ¹²Group ,-CR ¹⁶R ¹⁷C (O) NH ₂Group ,-C (=CR ²⁶R ²⁷) COR ¹⁰Group ,-C (=CR ²⁶R ²⁷) CO ₂R ²⁰Group ,-C (=CR ²⁶R ²⁷) P (O) (OR ¹⁰) ₂Group ,-C (=CR ²⁶R ²⁷) P (S) (OR ¹⁰) ₂Group ,-C (=CR ²⁶R ²⁷) C (O) NR ¹¹R ¹²Group ,-C (=CR ²⁶R ²⁷) C (O) NH ₂Group, or in the ring that Q-1 to Q-7 represents any one:

It can be replaced by at least a substituting group on ring, and described substituting group is selected from halogen atom, C ₁-C ₆Alkyl, C ₁-C ₆Haloalkyl, C ₂-C ₆Thiazolinyl, C ₂-C ₆Haloalkenyl group, C ₂-C ₆Alkynyl, C ₃-C ₆The halo alkynyl, C ₂-C ₈Alkoxyalkyl ,-OR ²⁸Group ,-SR ²⁸Group ,-NR ¹¹R ²⁸Group, C ₃-C ₈Alkoxycarbonyl alkyl, C ₂-C ₄Carboxyalkyl ,-CO ₂R ²⁸Group and cyano group;

R ¹¹And R ¹³Represent hydrogen atom or C independently ₁-C ₄Alkyl;

R ¹⁴Expression C ₁-C ₄Alkyl or the ring on can be with being selected from halogen atom, C ₁-C ₃Alkyl and C ₁-C ₃The phenyl that substituting group replaced of haloalkyl; Or,

R ¹⁹The expression hydrogen atom, C ₁-C ₄Alkyl or halogen atom;

R ²⁶And R ²⁷Can represent C with they bonded carbon atoms ₃-C ₈Cycloalkyl, or each ring that so forms can be with at least a halogen atom that is selected from, C ₁-C ₃Alkyl and C ₁-C ₃The substituting group of haloalkyl replaces; And

R ²⁸The expression hydrogen atom, C ₁-C ₆Alkyl, C ₁-C ₆Haloalkyl, C ₃-C ₆Thiazolinyl, C ₃-C ₆Haloalkenyl group, C ₃-C ₆Alkynyl, C ₃-C ₆The halo alkynyl, C ₂-C ₄Carboxyalkyl, C ₃-C ₈Alkoxy carbonyl alkyl, C ₃-C ₈The halo alkoxy carbonyl alkyl, C ₅-C ₉Thiazolinyl oxygen base carbonylic alkyl, C ₅-C ₉Haloalkenyl group oxygen base carbonylic alkyl, C ₅-C ₉Alkynyloxy base carbonylic alkyl, C ₅-C ₉Halo alkynyloxy base carbonylic alkyl, C ₅-C ₉Cyclo alkoxy carbonyl alkyl or C ₅-C ₉Halo cyclo alkoxy carbonyl alkyl.

32. a method of assessing cell to the resistance of formula (I) compound, described method comprises:

(1) with described compound with express according to the step of the proteinic cells contacting of claim 20 and

(2) be evaluated in the above-mentioned steps (1) step to the cells injury degree that contacts described compound.

33. according to the method for claim 32, wherein said cell is microorganism cells or vegetable cell;

34. select the method for the cell of anti-formula (I) compound, described method comprises the step of selecting cell in the method according to claim 32 based on the resistance of assessing.

35. by the cell of the antiweed selected according to the method for claim 34, or its culture.

36. a method of assessing plant to the resistance of formula (I) compound, described method comprises:

(1) with described compound with express according to the step of the proteinic plant contact of claim 20 and

(2) be evaluated in the above-mentioned steps (1) step to the degree of injury of the plant that contacts described compound.

37. a method of selecting the plant of anti-formula (I) compound, described method comprises the step of selecting plant in the method according to claim 36 based on the resistance of assessing.

38. one kind by the herbicide resistant plants of selecting according to the method for claim 37, or its offspring.

39. the method for a processing formula (I) compound, described method are included under the existence of the electron transport system that contains electron donor described compound and proteins react according to claim 20.

40. method according to claim 39, wherein by described compound is contacted with transformant described compound and described proteins react, in described transformant, the DNA introducing of code for said proteins can be made in the host cell of the position that it expresses in described cell.

41. the application of protein in processing formula (I) compound according to claim 20.

42. coding is according to the application of proteinic polynucleotide in processing formula (I) compound of claim 20.